Ceramidase gene

ABSTRACT

A neutral/alkaline ceramidase derived from a mammal; an antibody specifically binding thereto; a probe and primer which are capable of specifically hybridizing thereto; a method for producing the ceramidase by a genetic engineering means; a method for detecting the ceramidase or the gene; and a method of controlling an amount of a ceramide in a cell and/or in a tissue. The present invention is useful as a reagent for lipid engineering for analyzing a structure, functions, and the like of a ceramide, and in its applications to diseases associated with the ceramide metabolism.

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/JP00/01802 which has an Internationalfiling date of Mar. 24, 2000, which designated the United States ofAmerica and was not published in English.

TECHNICAL FIELD

The present invention relates to a polypeptide possessing a ceramidaseactivity, an antibody specifically binding thereto, a gene encoding thepolypeptide, and a probe and primer which are capable of specificallyhybridizing thereto, which are useful as a reagent for lipid engineeringfor analyzing a structure, functions, and the like of a ceramide. Inaddition, the present invention relates to a method for producing theabove-mentioned polypeptide by a genetic engineering means, and a methodfor detecting the polypeptide or the gene, and a kit therefor. Further,the present invention relates to a method of controlling an amount of aceramide in a cell and/or in a tissue, which can be applied to a diseasecaused by abnormality in the amount of the ceramide.

BACKGROUND ART

A ceramidase is an enzyme which hydrolyzes a ceramide, a kind of asphingolipid, into a sphingoid and a fatty acid. The sphingoid which isgenerated by hydrolyzing the ceramide with the ceramidase possessesvarious physiological activities such as inhibition of protein kinase C,activation of phospholipase D and inhibition of a calmodulin-dependentenzyme. As described above, the above-mentioned sphingoid is animportant substance which is thought to be acting on the regulation ofthe cell functions because the sphingoid is involved in proliferation ofcells and intracellular signal transduction. The ceramidase is an enzymewhich plays an important role of the control of the amount of theabove-mentioned sphingoid.

Ceramidases are classified into acidic ceramidases and neutral/alkalineceramidases by the optimum pH. There have been, so far reported that thepresence of a ceramidase possessing the optimum pH in an acidic rangehas been found in mammalian tissues such as rat brain [Biochemistry, 8,1692-1698 (1969)], guinea pig epithelial cells [J. Biol. Chem., 270,12677-12684 (1995)], human kidney [Biochim. Biophys. Acta, 398, 125-131(1975)], spleen [Biochim. Biophys. Acta, 1004, 245-251 (1989)],fibroblasts [Biochem. J., 205, 419-425 (1982)], and epithelium [FEBSLett., 268, 110-112 (1990)]; and human urine [J. Biol. Chem., 270,11098-11102 (1995)], and the like.

In addition, it has been clarified that a bacterium belonging to thegenus Pseudomonas produces a ceramidase, and this ceramidase is aceramidase possessing optimum pH in an alkaline region [J. Biol Chem.,273, 14368-14373 (1998)].

Among these ceramidases, amino acid sequences of the acidic ceramidasepurified from human urine and nucleotide sequences of a gene encodingthe ceramidase have been determined [J. Biol Chem, 271, 33110-33115(1996)]. In addition, an acidic ceramidase gene of a mouse has beenobtained by utilizing its homology with the above-mentioned acidicceramidase gene derived from human urine [Genomics, 50, 267-274 (1998)].

However, since all of the ceramidase genes derived from mammals whichhave been reported encode acidic ceramidases, amino acid sequences andgenomic structures of the neutral/alkaline ceramidase in mammals havebeen completely unknown, so that biological functions of theneutral/alkaline ceramidase in higher organisms have not yet beenelucidated at present.

In the studies on the elucidation of the in vivo functions of aceramide, metabolic control therefor, diagnosis or treatment of adisease associated with the ceramide or the like, it is necessary toobtain detailed information concerning various enzymes associated withthe ceramide, and the enzyme gene. However, as mentioned above, findingson the amino acid sequence and genes thereof of the neutral/alkalineceramidase in mammals have not yet been obtained at present. Therefore,in order to develop the technique as described above pertaining to aceramide, it is necessary to obtain some findings associated with aneutral/alkaline ceramidase, especially a gene thereof.

As mentioned above, although several reports have been made on cloningof ceramidase genes of a mammal, all of these reports are concerned withceramidases possessing an activity in an acidic region, which cannot beexpected to possess a homology with a ceramidase possessing an activityin a neutral/alkaline region. Therefore, it has been difficult to obtaina neutral/alkaline ceramidase gene as a homolog of a nucleotide sequenceof the acidic ceramidase gene.

DISCLOSURE OF INVENTION

The present invention has been accomplished in view of theabove-described prior art and a first object of the present invention isto provide a neutral/alkaline ceramidase gene of a mammal. A secondobject of the present invention is to provide a method for producing aneutral/alkaline ceramidase gene in a high purity by a geneticengineering means, comprising using a transformant resulting fromincorporation of an expression vector carrying the gene. A third objectof the present invention is to provide a polypeptide encoding theabove-mentioned gene. A fourth object of the present invention is toprovide an antisense DNA and an antisense RNA which are complementary tothe gene of the present invention or a part thereof. A fifth object ofthe present invention is to provide a synthetic oligonucleotide probe orprimer capable of specifically hybridizing to the gene of the presentinvention. A sixth object of the present invention is to provide anantibody or a fragment thereof specifically binding to the polypeptide.A seventh object of the present invention is to provide a method fordetecting the above-mentioned ceramidase or a gene thereof, and a kitused therefor. An eighth object of the present invention is to provide amethod of controlling an amount of ceramide in the cell or in thetissue.

The present inventors have succeeded in isolating a neutral/alkalineceramidase from liver of a mouse, a mammalian, and isolating the genes.In addition, they have succeeded in elucidating the structure of theneutral/alkaline ceramidase of a mammal including human by usingtechniques such as hybridization or polymerase chain reaction (PCR).Further, they have also succeeded in conveniently producing aneutral/alkaline ceramidase in a high purity by genetic engineeringtechniques by using the genes. Thus, the present invention has beenperfected thereby.

Specifically, the gist of the present invention relates to:

[1] a gene having a nucleotide sequence of a nucleic acid selected fromthe group consisting of:

(A) a nucleic acid encoding a polypeptide having the amino acid sequenceof SEQ ID NO: 14 of the Sequence Listing or a partial sequence thereof,the polypeptide possessing a ceramidase activity;

(B) a nucleic acid having a nucleotide sequence of SEQ ID NO: 15 of theSequence Listing or a partial sequence thereof and encoding apolypeptide possessing a ceramidase activity;

(C) a nucleic acid encoding a polypeptide consisting of an amino acidsequence resulting from deletion, addition, insertion or substitution ofat least one amino acid residue in the amino acid sequence of SEQ ID NO:14 of the Sequence Listing, the polypeptide possessing a ceramidaseactivity;

(D) a nucleic acid consisting of a nucleotide sequence resulting fromdeletion, addition, insertion or substitution of at least one base inthe nucleotide sequence of SEQ ID NO: 15 of the Sequence Listing andencoding a polypeptide possessing a ceramidase activity;

(E) a nucleic acid capable of hybridizing to a complementary strand of anucleic acid of any one of the above (A) to (D), under stringentconditions, and encoding a polypeptide possessing a ceramidase activity;and

(F) a nucleic acid having a nucleotide sequence different from thenucleic acid of any one of above (A) to (E) via degeneracy and encodinga polypeptide possessing a ceramidase activity;

[2] a recombinant DNA comprising the gene of item [1] above;

[3] an expression vector for a microorganism, an animal cell or a plantcell, comprising the gene of item [1] above or the recombinant DNA ofitem [2] above;

[4] a transformant carrying the expression vector of item [3] above;

[5] a method for producing a polypeptide possessing a ceramidaseactivity, characterized by culturing the transformant of item [4] aboveunder conditions appropriate for expression of the ceramidase gene andproduction of the polypeptide encoded by the gene, and collecting apolypeptide possessing a ceramidase activity from the resulting culture;

[6] a polypeptide having the amino acid sequence of SEQ ID NO: 14 of theSequence Listing or a partial sequence thereof and possessing aceramidase activity;

[7] a polypeptide possessing a ceramidase activity, encoded by the geneof item [1] above;

[8] an antisense DNA which is complementary to the gene of item [1]above or a part thereof;

[9] an antisense RNA which is complementary to the gene of item [1]above or a part thereof;

[10] an expression vector comprising the antisense DNA of item [8]above;

[11] an oligonucleotide probe or primer, capable of specificallyhybridizing to the gene of item [1] above or a complementary strandthereof;

[12] an antibody or a fragment thereof, capable of specifically bindingto the polypeptide of item [6] or item [7] above;

[13] a method for detecting a gene encoding a polypeptide possessing aceramidase activity, comprising using the oligonucleotide probe orprimer of item [11] above;

[14] a kit for the use in detection of a gene encoding a polypeptidepossessing a ceramidase activity, comprising the oligonucleotide probeand/or primer of item [11] above;

[15] a method for detecting a polypeptide possessing a ceramidaseactivity, comprising using the antibody or a fragment thereof of item[12] above;

[16] a kit for the use in detection of a polypeptide possessing aceramidase activity, comprising the antibody or a fragment thereof ofitem [12] above; and

[17] a method of controlling an amount of ceramide in a cell and/or in atissue, characterized by introducing the gene of item [1] above or anantisense nucleic acid thereof into the cell and/or the tissue, therebycontrolling the amount of ceramide in the cell and/or in the tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing optimum pH of the ceramidase.

FIG. 2 is a restriction endonuclease map of a DNA fragment comprisingthe ceramidase gene.

BEST MODE FOR CARRYING OUT THE INVENTION

Since information such as consensus sequences for a neutral/alkalineceramidase has not been clarified, the present inventors have obtainedthe amino acid information by isolating the above-mentionedneutral/alkaline ceramidase, whereby a gene encoding the above-mentionedceramidase could be isolated for the first time.

From the above findings, there could be obtained a surprising findingthat the amino acid sequence of the neutral/alkaline ceramidase derivedfrom a mouse liver of the present invention has a low homology with theamino acid sequence of a known alkaline ceramidase [previouslymentioned, J. Biol. Chem., 273, 14368-14373 (1998)] derived from abacterium belonging to the genus Pseudomonas (Pseudomonas aeruginosa).

The ceramidase of the present invention has been clarified for the firsttime as a neutral/alkaline ceramidase derived from a mammal. Therefore,the present invention is even more useful in the developments ofelucidation of the in vivo functions of a ceramide, metabolic controltherefor, and diagnosis and treatment of diseases associated with aceramide, and the like, as compared to the known alkaline ceramidasederived from the known bacterium of the genus Pseudomonas.

The present invention will be described hereinbelow.

(1) Polypeptide Possessing Ceramidase Activity

In the present specification, the phrase “polypeptide possessing aceramidase activity” (which may be simply referred to as “ceramidase” inthe present specification) refers to an enzyme possessing an activity ofhydrolyzing a ceramide to generate a sphigoid and a fatty acid asmentioned above. In addition, the term “neutral/alkaline ceramidase”refers to a ceramidase possessing an optimum pH at a pH higher than theacidic range.

As one example thereof, enzymologically chemical and physicochemicalcharacteristics of the isolated and purified, neutral/alkalineceramidase derived from a mouse liver in the present invention will bedescribed.

1. Action

The ceramidase of the present invention acts to hydrolyze a ceramide,thereby generating a sphingoid and a fatty acid.

The activity of the ceramidase can be determined in accordance with themethod described, for instance, in J. Biol. Chem., 275, 3462-3468(2000).

Concretely, a reaction mixture prepared by dissolving 550 pmol of12-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl)sphingosine,hereinafter referred to as C12-NBD-ceramide [Anal. Biochem., 263,183-188 (1998)], 1.0% (W/V) sodium cholate and a suitable amount of anenzyme (ceramidase) in 20 μl of 25 mM Tris-hydrochloric acid buffer (pH7.5) is incubated at 37° C. for 30 minutes. The reaction mixture isincubated in a boiling water bath for 5 minutes, thereby stopping thereaction. The resulting reaction mixture is further evaporated todryness under reduced pressure. The dried solid is dissolved inchloroform/methanol=2/1 (V/V), and developed by silica gel thin layerchromatography (developing solvent: chloroform/methanol/25% aqueousammonia=90/20/0.5 (V/V/V)). Thereafter, the12-(N-7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl acid,hereinafter referred to as C12-NBD-fatty acid, generated by theabove-mentioned reaction is quantified by using CS-9300 Chromatoscanner(manufactured by Shimadzu Corporation) at an excitation wavelength of475 nm and a fluorescent wavelength of 525 nm. One unit (U) of thisenzyme (ceramidase) is defined as an amount of the enzyme required forreleasing 1 micromol of the C12-NBD-fatty acid, per one minute under theabove-mentioned conditions, resulting from hydrolysis of theC12-NBD-ceramide.

2. Substrate Specificity

Five milliunits of the ceramidase of the present invention is acted on100 pmol of various kinds of sphingolipids, in which the fatty acidmoiety is labeled with ¹⁴C-radioactive isotope, in 20 ml of 25 mMTris-hydrochloric acid buffer, pH 7.5, containing 1% sodium cholate at37° C. for 24 hours. The reaction mixture is developed on silica gelthin layer chromatography. Thereafter, the ¹⁴C-labeled sphingolipids andthe ¹⁴C-labeled fatty acids generated by the enzymatic reaction aredetected and quantified by Imaging Analyzer BAS 1000 (manufactured byFuji Photo Film). The degradation ratio is calculated from the obtainedvalues. The substrate specificities of the ceramidase of the presentinvention are shown in Table 1.

As shown in Table 1, the ceramidase of the present invention showssubstrate specificities of: (1) hydrolyzing various N-acylsphingosines;(2) not acting on galactosyylceramide, sulfatide,Galb1-3GalNAcb1-4(NeuAca2-3)Galb1-4Glcb1-1′Cer (GM1a), or sphingomyelin;(3) acting favorably on a ceramide containing sphingenin (d18:1) than aceramide containing sphinganine (d18:0); (4) being less likely to act aceramide containing phytosphingosine (t18:0); and the like.

TABLE 1 Degradation Substrate (Structure) Ratio (%) N-Lauroylsphingosine(C12:0/d18:1) 63 N-Palmitoylsphingosine (C16:0/d18:1) 93N-Stearoylsphingosine (C18:0/d18:1) 83 N-Palmitoylsphinganine(C16:0/d18:0) 59 N-Stearoylsphinganine (C18:0/d18:0) 40N-Palmitoylphytosphingosine (C16:0/t18:0) 5 N-Stearoylphytosphingosine(C18:0/t18:0) 2 12-((N-(7-nitrobenz-2-oxa-1,3-diazol-4- 97 yl) amino)dodecanoyl) sphingosine (NBD-N- Dodecanoylsphingosine) (NBD-C12:0/d18:1)6-((N-(7-nitrobenz-2-oxa-1,3-diazole-4- 2 yl) amino) hexanoyl)sphingosine (NBD-N- Hexanoylsphingosine) (NBD-C6:0/d18:1)Galactosylceramide (Galb1-1′Cer) 0 Sulfatide (HSO3-3Galb1-1′Cer) 0GM1a(Galb1-3GalNAcb1-4 (NeuAca2-3)Galb1-4Glcb1- 0 1′Cer) Sphingomyelin(Choline phosphate Cer) 0

3. Optimum pH

Sixteen milliunits of the ceramidase of the present invention is actedon 100 pmol of C12-NBD-ceramide in 20 ml of 3,3-dimethylglutaric acid,50 mM Tris-(hydroxymethyl)aminomethane, 50 mM2-amino-2-methyl-1,3-propanediol at 37° C. for 24 hours. The resultingreaction mixture is developed by silica gel thin layer chromatography.Next, the NBD-labeled ceramide and the NBD-labeled fatty acid generatedby the enzymatic reaction are detected and quantified at a detectionwavelength of 525 nm by using the CS-9300 Chromatoscanner. Thedegradation ratio is calculated from the obtained values. FIG. 1 is agraph showing the relationship between the degradation activity of theC12-NBD-labeled ceramide and pH, wherein the ordinate axis shows thedegradation ratio (%), and the abscissa axis shows a reaction pH. Asshown in the results of FIG. 1, the optimum pH of the present ceramidaseis from 7.0 to 8.0.

4. Temperature Stability

The ceramidase of the present invention does not show the reducedactivity when treated in 20 mM Tris-hydrochloric acid (pH 7.5) buffercontaining 0.1% polidocanol [trade name: Lubrol PX] at 37° C. for 24hours, but shows the reduced activity by a treatment at 60° C. for 1hour, to about 30% of the activity before the treatment.

5. Molecular Weight

The molecular weight of the ceramidase of the present invention is about94 kDa on SDS-PAGE (under reducing conditions). In addition, the presentenzyme digested by glycopeptidase F is about 73 kDa on SDS-PAGE (underreducing conditions).

In the present specification, one example of the “polypeptide possessinga ceramidase activity” includes a polypeptide of a naturally occurringceramidase derived from a mouse, having the amino acid sequence of SEQID NO: 14 of the Sequence Listing. Further, not only the polypeptidehaving the amino acid sequence, but also a polypeptide having an aminoacid sequence with a mutation such as substitution, deletion, additionor insertion of one or more amino acids introduced into the amino acidsequence of SEQ ID NO: 14 of the Sequence Listing are encompassed in theterm “polypeptide possessing a ceramidase activity,” as long as thepolypeptide can be found to possess a similar ceramidase activity inaccordance with the activity determination by the method describedabove. In addition, in the above-mentioned mutation, two or more kindsof mutations may be introduced, as long as they are mutations by whichthe resulting polypeptide can exhibit a ceramidase activity. In thepresent specification, the term “amino acid sequence with a mutationintroduced” encompasses any of amino acid sequences into which amutation is artificially introduced and amino acid sequences having anaturally occurring mutation.

The ceramidase having the mutation can be concretely obtained, forinstance, by selecting a polypeptide possessing a ceramidase activityfor the mutated gene having a mutation in the nucleotide sequence (SEQID NO: 15) of the ceramidase gene described below, by the followingsteps:

(a) incubating a gene expression product in a reaction mixture[composition: 550 pmol of C12-NBD-ceramide and 1.0% (W/V) sodium cholatein 20 μl of 25 mM Tris-hydrochloric acid buffer (pH 6 to 9, preferably6.5 to 8.5, more preferably 7 to 8, especially preferably 7.5)] at 37°C. for 30 minutes, to react the mixture; and

(b) detecting generation of a C12-NBD-fatty acid in the reactionproduct.

(2) Ceramidase Gene

The ceramidase gene in the present invention refers to a gene having anucleotide sequence of a nucleic acid encoding the above-mentioned“polypeptide possessing a ceramidase activity,” or a nucleic acidcomprising a nucleotide sequence of the gene. One example thereofincludes a gene having a nucleotide sequence of a nucleic acid encodinga polypeptide consisting of the amino acid sequence of SEQ ID NO: 14 ofthe Sequence Listing or a partial sequence thereof; and a gene havingthe nucleotide sequence of a nucleic acid consisting of a nucleotidesequence of SEQ ID NO: 15 of the Sequence Listing, or a partial sequencethereof, and the present invention is not limited to these exemplified.As described above, even a gene having a nucleotide sequence of anucleic acid encoding a polypeptide consisting of a partial sequence ofthe amino acid sequence of SEQ ID NO: 14, or a gene having a nucleotidesequence of a nucleic acid consisting of a partial sequence of thenucleotide sequence of SEQ ID NO: 15 are encompassed in the scope of thepresent invention, as long as the gene encodes a polypeptide possessinga ceramidase activity. These genes are neutral/alkaline ceramidase genesderived from mouse liver, and the origin of the ceramidase gene of thepresent invention is not particularly limited, as long as the ceramidaseactivity of the gene product can be detected in the same manner as inthe steps (a) and (b) described in item (1) above. The origin includes,for instance, mice, rats, humans, hamsters, guinea pigs, and the like.

In the present specification, the phrase “polypeptide consisting of apartial sequence (of the amino acid sequence)” means those in which theceramidase activity can be detected by the steps (a) and (b) describedin item (1) above.

Further, a gene encoding a ceramidase having a mutation, which canexhibit a similar ceramidase activity, is also encompassed in thepresent invention. For instance, even a gene having a nucleotidesequence of a nucleic acid encoding an amino acid sequence with amutation such as deletion, addition, insertion or substitution of one ormore amino acid residues introduced into the amino acid sequence of SEQID NO: 15 of the Sequence Listing is encompassed in the gene of thepresent invention, as long as the polypeptide encoded by the genepossesses a ceramidase activity. Even a naturally occurring gene havinga mutation as well as an artificially prepared gene described above areencompassed in the scope of the present invention, as long as the geneis a gene having a nucleotide sequence of a nucleic acid encoding apolypeptide possessing an activity for a neutral/alkaline ceramidase.

As the method for preparing a gene into which a mutation is artificiallyintroduced as described above, for instance, the following method isused.

As a method of introducing a random mutation, there can be employed, forinstance, a method for generating a transition mutation in whichcytosine base is substituted by uracil base with a chemical treatmentusing sodium hydrogen sulfite [Proceedings of the National Academy ofSciences of the USA, 79, 1408-1412 (1982)]; a method for lowering anaccuracy of incorporation of nucleotide during DNA synthesis by carryingout PCR in a reaction mixture containing manganese [Anal. Biochem., 224,347-353 (1995)]; and the like.

As a method for introducing site-directed mutagenesis, there can beemployed, for instance, a method utilizing amber mutation [gapped duplexmethod, Nucleic Acids Research, 12, 9441-9456 (1984)]; a method ofutilizing a dut (dUTPase) gene and ung (uracil-DNA glycosilase) genedeficient host [Kunkel method, Proceedings of the National Academy ofSciences of the USA, 82, 488-492 (1985)]; a method by PCR utilizingamber mutation (WO 98/02535); and the like. Various kinds of kits forintroducing site-directed mutagenesis to a desired gene by these methodsare commercially available, and a gene resulting from introduction of adesired mutation can be readily obtained by utilizing the kits.

In addition, the nucleic acid capable of hybridizing to a complementarystrand of the above-mentioned nucleic acid under stringent conditions,the gene having a nucleotide sequence of a nucleic acid encoding apolypeptide possessing a ceramidase activity is also encompassed in thegene of the present invention. The ceramidase activity can be detected,for instance, by the steps (a) and (b) described in item (1) above.

Here, the term “stringent conditions” refers to, for instance, thefollowing conditions. Concretely, the term refers to conditions ofcarrying out incubation at 50° C. for 4 hours to overnight in 6×SSC(wherein 1×SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0)containing 0.5% SDS, 5×Denhardt's [0.1% bovine serum albumin (BSA), 0.1%polyvinyl pyrrolidone, 0.1% Ficol 400], and 100 μg/ml salmon sperm DNA.

Also, the details of the hybridization manipulations are described, forinstance, in Molecular Cloning: A Laboratory Manual, 2nd Ed., publishedby Cold Spring Harbor Laboratory in 1989, edited by T. Maniatis et al.

The gene having a nucleotide sequence different from the nucleic acidmentioned above via a degenerated genetic code is also encompassed inthe ceramidase gene of the present invention.

The polypeptide encoded by the gene of the present invention isencompassed in the present invention, as long as its ceramidase activitycan be detected by the steps (a) and (b) described in item (1) above.

According to the gene of the present invention, there is furtherprovided a recombinant DNA suitable for various purposes used. Here, theterm “recombinant DNA” refers to a DNA carrying the gene of the presentinvention, obtained by genetic engineering technique.

The recombinant DNA carrying the ceramidase gene of the presentinvention is ligated to a known vector or the like, whereby anexpression vector inserted with the ceramidase gene in an expressiblestate can be prepared. Such an expression vector is also encompassed inthe present invention.

In the present invention, an expression vector refers to a vector whichis constructed such that the gene or recombinant DNA mentioned above isinserted and expressed in desired host cells. In addition, a vector inwhich an antisense DNA as described below is inserted is alsoencompassed in the expression vector of the present invention. Thevector to be inserted includes plasmid vectors, phage vectors, viralvectors, and the like. As the plasmid vector, commercially availableproducts such as pUC18, pUC19, pBluescript and pET can be suitably used,and as the phage vectors, commercially available products of lambdaphage vectors such as λgt10 and λgt11 can be suitably used, withoutbeing limited thereto. As the viral vectors, retroviral vector,adenoviral vector, vaccinia viral vector, adeno-associated viral vector,and the like can be used, without being limited thereto. These vectorsare appropriately selected in accordance with the host cells used. Eachof these vectors may appropriately carry a factor such as an induciblepromoter, a selectable marker gene, or a terminator.

In addition, in order to facilitate isolation and purification, a vectorcarrying a sequence capable of expressing as His tag or GST fusionprotein may be used depending upon its use. In this case, GST(glutathione S-transferase) fusion protein vector carrying anappropriate promoter (for instance, lac, tac, trc, trp, CMV, SV40 earlypromoter, or the like) which functions in a host cell (for instance,pGEX4T), a vector carrying a tag (for instance, Myc, HisA, or the like)sequence or the like can be used.

(3) Transformant Harboring Ceramidase Gene

The transformant of the present invention, namely the cells capable ofexpressing the ceramidase gene of the present invention, can be obtainedby transforming a host with an expression vector in which the ceramidasegene of the present invention is inserted. The host-used can beappropriately selected depending upon the purposes used of the desiredceramidase, and microorganisms such as Escherichia coli, yeasts, animalcells, plant cells, animal individuals, plant individuals, and the like.Concretely, Escherichia coli includes HB101 strain, C600 strain, JM109strain, DH5α strain, DH10B strain, XL-1BlueMRF′ strain, TOP10F strainand the like of the Escherichia coli K-12 derivative. Also, the yeastcells include Saccharomyces cerevisiae and the like. The animal cellsinclude L, 3T3, FM3A, CHO, COS, Vero, Hela, and the like. The plantcells include tobacco BY2 and the like.

As a method for transducing an expression vector into a host, a method,including, for instance, described in Molecular Cloning: A LaboratoryManual, 2nd Ed., 249-254 may be employed. Next, in order to select atransformant expressing a desired gene, the characteristics of theexpression vector are utilized. For instance, in a case where a plasmidvector is pBluescript and a host cell Escherichia coli, a colony havingampicillin resistance on a plate containing ampicillin is selected, or acolony having ampicillin resistance and showing white color on a platecontaining ampicillin, 5-bromo-4-chloro-3-indolyl-β-D-galactoside(X-Gal) and isopropyl-β-D-thiogalactopyranoside (IPTG) is selected,thereby selecting a colony in which a foreign gene is transduced.

The polypeptide possessing a ceramidase activity can be produced byculturing the transformant of the present invention under generallyemployed conditions. In some cases, the codon usage differs dependingupon a host in which the gene of the present invention is expressed, sothat expression is suppressed. In this case, the codon used in the geneof the present invention may be changed to a codon suitable to eachhost. In addition, the above-mentioned expression vector is not limitedonly to those vectors derived from plasmids, and vectors derived fromphages, cosmids, and the like may also be used, as long as the object ofthe present invention is not hindered. A vector capable of inducing andexpressing a foreign gene, a vector capable of expressing as a fusionprotein with a reporter gene product, and the like are desirable, fromthe viewpoint of readily and massively producing the polypeptide of thepresent invention.

The expression of the ceramidase can be confirmed by determining theceramidase activity. The activity can be determined by, for instance,the method described in J. Biol. Chem., 275, 3462-3468 (2000) using acell extract of the transformant as a sample, for instance, similarprocedures to the steps (a) and (b) described in item (1) above. Inaddition, the expression of the ceramidase can be confirmed bydetermining the amount of a ceramide in the cell. The above-mentionedamount of ceramide can be determined, for instance, by the methoddescribed in Analytical Biochemistry, 244, 291-300 (1997). Also, anantibody against the ceramidase can be used. In a case where theceramidase is expressed as a fusion body with another polypeptide (thepolypeptide excluding the ceramidase of the present invention), anantibody against this polypeptide moiety, the polypeptide excluding theceramidase of the present invention, may be used. In a case where anantibody is used, the ceramidase can be detected by, for instance,subjecting a cell extract of the transformant to electrophoresis on anSDS-polyacrylamide gel, and thereafter transferring the electrophoresedgel on a polyvinylidene fluoride (PVDF) membrane, and detecting with theantibody on this membrane.

(4) Method for Producing Polypeptide Possessing Ceramidase Activity

The present invention also provides a method for producing a polypeptidepossessing a ceramidase activity, comprising the steps of culturing theabove-mentioned transformant under conditions appropriate for expressionof the ceramidase gene of the present invention and production of thepolypeptide encoding the gene, and collecting a polypeptide possessing aceramidase activity from the resulting culture. The method for culturingthe transformant is not particularly limited, and an appropriate one canbe selected from a known culturing method appropriate for the host used.

In the production method of the present invention, in a case where theabove-mentioned transformant is a microorganism or a cultured cell, theceramidase can be efficiently produced by determining optimum conditionsfor expression of the ceramidase in a medium composition, a pH of amedium, a culturing temperature, and a culturing time, as well as theamount of an inducer used, and the using time period, and the like.

A general method is employed in the purification of the ceramidase fromthe culture of the transformant. In a case where the transformantintracellularly accumulates the ceramidase as in the case of Escherichiacoli, after the termination of culture, the transformant cells areharvested by centrifugation, and the resulting cells are disrupted bysonication or the like, and thereafter centrifuged or the like to give acell-free extract. The cell-free extract used as a starting material canbe purified by salting-out as well as a general protein purificationmethod such as various kinds of chromatographies such as ion-exchangechromatography, gel filtration chromatography, hydrophobicchromatography, and affinity chromatography. The expression product maybe extracellularly secreted depending upon the host-vector system usedin some cases. In such a case, the purification can be similarly carriedout from culture supernatant.

According to the method of the present invention, in a case where theceramidase is produced intracellularly, a desired ceramidase may coexistintracellularly with impurities such as various kinds of enzymes andproteins. Since the impurities are present in a trace amount as comparedto the amount of the expressed ceramidase, there is an excellentadvantage that its purification is extremely facilitated. In addition,in a case where an extracellular secretion-type vector is used as avector, the ceramidase is extracellularly secreted, so that the mediumcomponent or the like coexists in the fraction comprising theceramidase. However, since the fraction usually contains almost noprotein component which would hinder the ceramidase purification, thereis an excellent advantage in that, for instance, complicated separationand purification procedures which have been required in the purificationof the ceramidase from a mouse liver are not necessitated

In addition, in a case of a ceramidase derived from a fungus, there is apossibility that the enzyme itself has a sugar chain. A polypeptidepossessing a ceramidase activity and having no sugar chain can beproduced by using as a host cell a cell which does not possess a sugarchain-synthesizing ability, for instance, a mutant cell which has lost asugar chain-synthesizing ability of a prokaryote such as Escherichiacoli, Bacillus substilis or Actinomyces, or an yeast, a fungus, ananimal cell, an insect cell and a plant cell. Further, an enzyme havinga sugar chain can be produced. In this case, a polypeptide possessing aceramidase activity and having a sugar chain can be produced by using asa host cell a cell which possesses a sugar chain-synthesizing ability,for instance, an yeast, a fungus, an animal cell, an insect cell and aplant cell.

Also, the expression product may form an insoluble inclusion bodydepending upon the host-vector system used. In this case, after thetermination of culture, the transformant cells are harvested bycentrifugation, and the resulting cells are disrupted by sonication orthe like, and thereafter centrifuged or the like, thereby harvesting aninsoluble fraction containing the inclusion body. After washing theinclusion body, the inclusion body is solubilized with an generally usedprotein solubilizing agent, for instance, urea, guanidine hydrochloride,or the like, and purified by various kinds of chromatographies such asion-exchange chromatography, gel filtration chromatography, hydrophobicchromatography, and affinity chromatography as occasion demands.Thereafter, refolding procedures employing dialysis or dilution methodare carried out, whereby a preparation comprising a polypeptidepossessing a ceramidase activity can be obtained. If this preparation isfurther purified by various kinds of chromatographies as occasiondemands, a polypeptide possessing a ceramidase activity in a high puritycan be obtained.

(5) Probe for Hybridization and Primer for PCR

The oligonucleotide probe or primer of the present invention is capableof specifically hybridizing to the gene of the present invention, or acomplementary strand thereof. The oligonucleotide probe or primer isdesigned on the basis of the nucleotide sequence of the ceramidase geneof the present invention. For instance, the oligonucleotide probe orprimer can be prepared by chemical synthesis by a general method. Thenucleotide sequence for the oligonucleotide probe is not particularlylimited. The nucleotide sequence is those capable of hybridizing to theabove-mentioned ceramidase gene, or a nucleic acid having a nucleotidesequence complementary to the gene under stringent conditions. Theabove-mentioned term “stringent conditions” is not particularly limited.For instance, the term “stringent conditions” refers to conditions ofincubating overnight at a temperature of [(Tm of the above-mentionedprobe)—25° C.] in a solution containing 6×SSC, 0.5% SDS, 5×Denhardt's,and 100 mg/ml salmon sperm DNA, and the like. Also, the nucleotidesequence of the above-mentioned primer is not particularly limited, aslong as the primer is annealed to the above-mentioned ceramidase gene orthe gene having a nucleotide sequence complementary to the gene underusual reaction conditions for PCR so that the extension reaction by theDNA polymerase can be initiated.

Tm of the oligonucleotide probe or primer can be calculated, forinstance, by the following equation:

Tm=81.5−16.6(log₁₀[Na⁺])+0.41(% G+C)−(600/N)

wherein N is a chain length of the oligonucleotide probe or primer; and% G+C is a content of guanine and cytosine residues in theoligonucleotide probe or primer.

In addition, when the chain length of the oligonucleotide probe orprimer is shorter than 18 bases, Tm can be deduced from a product of thecontents of A+T (adenine+thymine) residues multiplied by 2° C., with asum of a product of the contents of G+C residues multiplied by 4° C.[(A+T)×2+(G+C)×4].

The chain length of the above-mentioned oligonucleotide probe is notparticularly limited. It is preferable that the chain length is 15 basesor more, more preferably 18 bases or more, from the viewpoint ofpreventing nonspecific hybridization.

In addition, as the primer of the present invention, there can be citedthe nucleic acids having the same nucleotide sequences as those for theabove-mentioned oligonucleotide probe. For instance, the primer can beprepared by, for instance, designing on the basis of the nucleotidesequence of the gene of the present invention, and chemicallysynthesizing it, and the like. The chain length of the primer is notparticularly limited. For instance, the primer having a chain length of15 to 40 bases can be used, especially one having a chain length of 17to 30 bases can be suitably used. The above-mentioned primer can be usedfor various gene amplification methods such as PCR method, whereby theceramidase gene of the present invention can be detected.

Also, as the above-mentioned oligonucleotide probe or primer, there maybe used a nucleic acid obtained by fragmenting a nucleic acid encoding anaturally occurring ceramidase by an enzymatic treatment such asendonuclease treatment or exonuclease treatment, a physical treatmentsuch as sonication, or the like, and subjecting the resulting fragmentto separation and purification by various kinds of nucleic acidseparation methods represented by agarose gel or the like. It is desiredthat the nucleic acid obtained as described above is derived from aregion having a sequence characteristic of the ceramidase.

Further, in order to more readily detect the nucleic acid to bedetected, the above-mentioned oligonucleotide probe or primer can besubjected to appropriate labeling in accordance with a known method tobe used in the detection of the ceramidase gene of the presentinvention. The labeling is not particularly limited. The oligonucleotideprobe or primer may be labeled with radioisotopes as well as variouslabels represented by fluorescent substances, and ligands such as biotinand digoxigenin.

A DNA having high homology to the ceramidase gene of the presentinvention can be cloned by screening a genomic DNA or cDNA derived froman organ other than a mouse liver or from an organism other than amouse, or a genomic DNA library or cDNA library, by using the probe forhybridization of the present invention.

In addition, a DNA fragment having high homology to the ceramidase geneof the present invention can be detected by PCR method from a genomicDNA or cDNA derived from an organ other than mouse liver or derived froman organism other than mouse, or a genomic DNA library or cDNA library,by using the primer of the present invention, and further its fulllength gene can also be obtained.

(6) Method for Detecting Gene

One of the big features of the method for detecting a gene of thepresent invention resides in that the gene in the sample to be detectedis detected by using the above-mentioned oligonucleotide probe and/orprimer.

In the method for detection of the present invention, the gene may bedetected by hybridization method or the like by using theabove-mentioned oligonucleotide probe, or the gene may be detected byDNA amplification method such as PCR method by using the above-mentionedprimer.

In a case of hybridization using the oligonucleotide probe, the samplesto be detected include, for instance, samples such as colonies andcultured cells of microorganisms, and tissue fragments, those obtainedby immobilizing DNA or RNA in these samples onto a membrane, DNA or RNAextracted from these samples, and the like.

The hybridization can be carried out in accordance with a known methoddescribed in Molecular Cloning: A Laboratory Manual, 2nd Ed. and thelike. The conditions for the hybridization can be appropriatelydetermined by the Tm value of the probe used, the GC content of thetarget DNA, and the like. For instance, the conditions described inMolecular Cloning: A Laboratory Manual, 2nd Ed. mentioned above or thelike can be applied.

In a case where the gene is detected by using the primer, the samples tobe detected include, for instance, microorganism samples such as cultureof microorganisms, colonies of microorganisms and bacterial cells ofmicroorganisms; samples derived from a living body such as culturedcells, tissues and tissue fragments; and the like. As these samples, forinstance, isolated microorganisms and cultured cells may be used in theoriginal states, or after being subjected to an appropriate treatment.In addition, solid samples such as tissues can be used by preparing anexudate or suspension. Also, supernatant of these samples, or thosesamples prepared by subjecting these samples to a cytolytic treatmentsuch as a treatment with a surfactant or supernatant thereof can beused. Further, the sample may be subjected to a procedure of removingother components in the sample within a range so as not to impair thenucleic acid to be detected.

In a case where the detection is carried out by PCR method by using theabove-mentioned primer, the PCR conditions can be appropriately selectedin accordance with the Tm value of the primer used, the length of theregion to be amplified and detected, and the like. In PCR, the desiredgene can be detected by confirming the presence or absence of theamplified product. The method for confirming the presence or absence ofamplification is not particularly limited. For instance, theconfirmation can be made by, for instance, subjecting a reaction mixturefor nucleic acid amplification to agarose gel electrophoresis, stainingthe gel with an appropriate nucleic acid staining reagent, such asethidium bromide or SYBER Green I, subjecting the gel to irradiationwith ultraviolet rays, and detecting the presence or absence of theband. The detection of the band may be observed by naked eyes, or thedetection can be made, for instance, by using a fluorescent imageanalyzer or the like.

In the method for detecting the gene of the present invention, in orderto increase the detection sensitivity, the above-mentioned probe andprimer may be used together. For instance, the gene can be detected inhigh sensitivity and accurately by amplifying the ceramidase geneexisting in a trace amount in the sample by PCR method with theabove-mentioned primer, and thereafter hybridizing the gene with theprobe.

In a case where the ceramidase gene is detected by the method fordetection of the present invention and the amount of the gene is furtherdetermined, the amount of the gene can be determined by quantifying anintensity of the signal ascribed to the hybridized probe, a fluorescentintensity of the band ascribed to a product amplified with the primer,or the like. The expression amount of the desired gene can be examinedby quantifying the amount by using mRNA as an object to be determined.

In addition, in the method for detection of the present invention, thedetection can be carried out more conveniently by using the kit for theuse in detection of the gene of the present invention. Such a kit isalso encompassed in the present invention. One of the features of theabove-mentioned kit resides in that the kit comprises theabove-mentioned oligonucleotide probe and/or the above-mentioned primer.The above-mentioned kit may contain various components used in thedetection procedures. For instance, in a case of a kit comprising anoligonucleotide probe, there may be contained various kinds of reagentsfor hybridization representatively exemplified by a membrane forimmobilizing a nucleic acid, a hybridization buffer, and the like. Also,in a case of a kit comprising a primer, there may be contained reagentsfor PCR representatively exemplified by thermostable DNA polymerases,dNTP mixed solutions, buffers for PCR, and the like. Further, there maybe contained reagents for detecting a probe or an amplified DNA, mediafor proliferating microorganisms, media for culturing cells, reagentsfor extracting nucleic acids from a sample, and the like.

(7) Antibody or Fragment Thereof Specifically Binding to PolypeptidePossessing Ceramidase Activity

The antibody or a fragment thereof specifically binding to thepolypeptide of the present invention is not particularly limited, aslong as the antibody or a fragment thereof possesses an ability ofspecifically binding to the polypeptide. The antibody may be any ofpolyclonal antibodies and monoclonal antibodies. Further, antibodiesmodified by known techniques or antibody derivatives, for instance,humanized antibodies, Fab fragments, single-chain antibodies, and thelike, can also be used The antibody of the present invention can bereadily prepared by appropriately immunizing a rabbit, a rat or a mouseusing all or a part of the polypeptide of the present invention inaccordance with the method described in, for instance, Current Protocolsin Immunology, edited by John E. Coligan, published by John Wiely &Sons, Inc., 1992. The antibody thus obtained is purified and thereaftertreated with a peptidase or the like, to give an antibody fragment. Inaddition, the antibody can be prepared by genetically engineering means.Further, the antibody or a fragment thereof of the present invention maybe subjected to various modifications in order to facilitate thedetection by enzyme immunoassay, fluoroimmunoassay, luminescentimmunoassay, or the like.

The antibody or a fragment thereof mentioned above encompasses thosewhich are capable of specifically binding to a certain partial fragmentof the polypeptide.

The use of the resulting antibody or a fragment thereof includesapplications to detection of ceramidase-producing bacteria, detection ofceramidase-expressed cell lines, detection of ceramidase proteins in thecultured cell or in the tissue, affinity chromatography, screening of anexpression product of various kinds of libraries (genomic DNA or cDNA),pharmaceuticals, diagnostic agents, reagents for researches, and thelike.

(8) Method for Detecting Polypeptide

A feature of the method for detecting the polypeptide of the presentinvention resides in that the polypeptide possessing a ceramidaseactivity is detected by the antibody or a fragment thereof mentionedabove.

In the present invention, as the samples to be detected, there can beused, for instance, cultures of microorganisms and animal cells, tissuefragments, cell disruptions of microorganisms and animal cells, extractsor washings of tissues such as skins, and protein samples such asmembranes immobilized with proteins derived from microorganisms, animalcells and tissues.

As to the detection of the specific binding of the antibody or afragment thereof to the above-mentioned polypeptide, a known method canbe utilized, and such a method includes, for instance, enzymeimmunoassay, fluoroimmunoassay, luminescent immunoassay, and the like.

In the method for detecting the polypeptide of the present invention,the detection can be more conveniently carried out by using the kit forthe use in the detection of the polypeptide of the present invention.Such a kit is also encompassed in the present invention. A feature ofthe above-mentioned kit resides in that the kit comprises theabove-mentioned antibody or a fragment thereof. In addition, the kit maycontain a reaction buffer, a labeled secondary antibody, a developingreagent, and the like.

(9) Antisense DNA and Antisense RNA

In the present invention, each of the terms “antisense DNA” and“antisense RNA” refers to those having a nucleotide sequencecomplementary to the ceramidase gene of the present invention or a partthereof, which suppresses or controls expression (transcription,translation) of the genetic information from the gene by forming adouble strand with an endogenous ceramidase gene (genomic DNA and mRNA).The length of the antisense DNA or antisense RNA can be changeddepending upon the specificity of the nucleotide sequence and the methodfor transducing the nucleotide sequence into a cell. The antisense DNAor antisense RNA can be prepared by artificially synthesizing with asynthesizer; expressing a gene in an opposite direction (direction ofantisense) of the usual direction by an enzymatic reaction with the geneof the present invention as a template; or the like. In a case whereexpression of the antisense RNA is desired in a living body, anexpression vector ligated to the gene of the present invention in anopposite direction of the usual direction is constructed, and theresulting expression vector may be transduced into a living body.

For instance, numerous antisense techniques such as suppression ofproliferation of HIV utilizing tat gene [Nucleic Acids Research, 19,3359-3368 (1991)] or rev gene [Proceedings of the National Academy ofSciences of the USA, 86, 4244-4248 (1989)] have been known. Therefore,according to these methods, expression of the endogenous ceranudase genecan be suppressed or controlled by using the antisense DNA or antisenseRNA of the present invention. In addition, the antisense DNA orantisense RNA of the present invention can be utilized as a researchreagent for in situ hybridization or the like.

(10) Control of Amount of Ceramide in Cell or in Tissue Using CeramidaseGene or Antisense Nucleic Acid Thereof

By the gene of the present invention, a method of controlling an amountof a ceramide in a cell and/or in a tissue can be further provided. Sucha method of control is also encompassed in the present invention. One ofthe features of the method of controlling an amount of a ceramide in acell and/or in a tissue of the present invention resides in that theceramidase gene of the present invention is introduced into the celland/or into the tissue, thereby controlling the amount of a ceramide inthe cell and for in the tissue.

Concretely, in the method of control of the present invention, theceramidase gene of the present invention is introduced into the celland/or into the tissue, and the ceramide is degraded in a cell or in atissue by an action of the ceramidase expressed by the gene. On theother hand, the ceramidase gene of the present invention is introducedinto the cell or into the tissue so as to generate the antisense nucleicacid of the gene, for instance, an antisense RNA, whereby the ceramidaseactivity in the cell or in the tissue is lowered, so that thedegradation of the ceramide can be suppressed. In addition, in a casewhere the amount of the ceramide is suppressed, the antisense nucleicacid of the ceramidase gene of the present invention, namely theantisense DNA or antisense RNA of the present invention, may beintroduced in a cell or in a tissue in an intact form.

As the method for introducing the ceramidase gene or an antisensenucleic acid thereof mentioned above in a cell or in a tissue, a knownmethod can be used, and methods for physically introducing a gene suchas electroporation method and particle gun method, or a method forintroducing a gene using a viral vector can be utilized. The viralvector which can be used in the method of the present invention is notparticularly limited. For instance, the gene can be introduced by usingretroviral vector, adenoviral vector, vaccinia viral vector,adeno-associated viral vector, or the like.

According to the method of control of the present invention, theceramidase gene or an antisense nucleic acid thereof of the presentinvention is introduced in a cell or in a tissue to control the amountof a ceramide in the cell and/or in the tissue, whereby exhibitingexcellent effects such that a disease caused by an abnormal amount of aceramide can be treated, and that a model animal suffering from adisease with abnormal ceramide metabolism can be prepared. The “diseasecaused by an abnormal amount of a ceramide” is not particularly limited,and includes, for instance, Farber's disease and the like.

The method for obtaining a ceramidase gene derived from a mouse liverwill be explained hereinbelow.

1) First, a membrane fraction is prepared from a homogenate of a mouseliver, and suspended in a sucrose-EDTA solution, and the suspension isfrozen and thawed, and thereafter centrifuged, to give supernatant(crude enzyme extract). A ceramidase preparation homogeneously purifiedcan be obtained from the resulting crude enzyme extract by combiningknown protein purification methods, for instance, various kinds ofchromatographies. As the chromatographies which can be used in theabove-mentioned purification, anion exchange chromatography, hydrophobicchromatography, chelating chromatography, gel filtration chromatography,and the like can be used.

2) Next, as the information for preparing a probe for cloning theceramidase gene, the partial amino acid sequences of the ceramidase areexamined. The N-terminal amino acid sequence of the ceramidase can befound by subjecting the above-mentioned purification ceramidasepreparation itself to amino acid sequencing by Edman degradation method[J. Biol. Chem., 256, 7990-7997 (1981)]. In addition, the partial aminoacid sequence of the internal ceramidase can be obtained by purifying anappropriate peptide fragment from a peptide mixture resulting fromdigestion of the purified enzyme preparation with a protease having ahigh substrate specificity, for instance, lysylendopeptidase,N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK)-trypsin, and the like,and subjecting the resulting fragment to amino acid sequencing.

3) On the basis of the information of the amino acid sequence thusclarified, an oligonucleotide to be used for the probe forhybridization, or used for the primer for PCR is designed, to clone theceramidase gene of the present invention. In order to carry the cloning,PCR or hybridization method generally employed is utilized. PCR methodcan be carried out in accordance with the method described in PCRTechnology, edited by Erlich, H. A., published by Stockton Press, 1989.The hybridization method can be carried out, for instance, in accordancewith a known method described in Molecular Cloning: A Laboratory Manual,2nd Ed.

4) As to the DNA fragment obtained by the hybridization or PCR mentionedabove, by decoding its nucleotide sequence, the amino acid sequencewhich can be encoded therein can be known. Whether or not theabove-mentioned DNA fragment is a fragment of the ceramidase gene can beconfirmed by comparing the sequence with the partial amino acid sequenceof the ceramidase obtained in item 2) above.

5) In a case where the DNA fragment obtained by hybridization or PCR initem 3) is a part of the ceramidase gene, a DNA fragment comprising agene encoding a full length ceramidase can be obtained by repeating theprocedures of item 3), or alternatively, preparing a new probe or primeron the basis of the nucleotide sequence of the DNA fragment obtained initem 3), and carrying out hybridization or PCR by using the probe orprimer.

6) An expression vector is constructed by ligating the gene encoding afull length ceramidase thus obtained with an appropriate vector, andthen the transformant in which the expression vector is transduced isprepared. The transformant is cultured, and the ceramidase activity inthe resulting culture is examined, whereby confirmation can be made suchthat the resulting gene is one encoding the ceramidase.

However, in the cloning of the ceramidase gene of the present invention,a probe DNA appropriate for screening of the library by hybridizationmethod could not be designed on the basis of the partial amino acidsequence information obtained in the present invention. In addition,although various PCR primers were designed by designing from each of thepartial amino acid sequences and the nucleotide sequences of vector forthe use in the library preparation, and the resulting primers were usedin various combination to carry out PCR, specific amplification was notfound, the only amplification being found alone in the combination oftwo kinds of primers designed on the basis of the partial amino acidsequence C-53 (shown in the amino acid sequence of C-53 in SEQ ID NO: 3of Sequence Listing). However, the amplified DNA fragment P-1 (shown inthe nucleotide sequence of P-1 in SEQ ID NO: 6 of Sequence Listing) wasas short as 68 bp, so that the amplified fragment itself could not beused as a probe for library screening by hybridization method.Therefore, a gene fragment of a size of 335 bp, which is thought to beappropriate for the first time as a probe for library screening byhybridization method, has been successfully obtained for the first timeby further designing a primer for PCR on the basis of the sequence ofP-1, and at the same time carrying out PCR in combination of the primerand a primer designed on the basis of the nucleotide sequence of avector used in the preparation of the library.

Further, a gene encoding a full length ceramidase can be cloned byscreening cDNA library derived from a mouse liver with theabove-mentioned fragment of a size of 335 bp as a probe. Also, thegenomic DNA of a ceramidase of the present invention can be obtained byscreening a genomic DNA library derived from a mouse liver.

The entire nucleotide sequence of the produced ceramidase gene from amouse liver thus obtained is shown in SEQ ID NO: 12 of Sequence Listing,and the amino acid sequence of the polypeptide encoded thereby is shownin SEQ ID NO: 13 of Sequence Listing. It has been found that the enzymeis processed to a mature enzyme in which the N-terminal part of thepeptide of the gene is removed in vivo on the bases of this amino acidsequence and the N-terminal amino acid sequence of the gene. The aminoacid sequence of this mature ceramidase and the nucleotide sequenceencoded by the sequence are shown in SEQ ID NOs: 14 and 15 of SequenceListing, respectively. Each of the above-mentioned amino acid sequenceand nucleotide sequence does not have homology with each of the knownamino acid sequence and nucleotide sequence of a ceramidase derived froma mammal. In other words, the ceramidase gene provided by the presentinvention consists of a completely novel sequence, which is irrelevantto the known ceramidase gene.

As described above, according to the present invention, there areprovided a primary structure and a genetic structure of the ceramidasederived from a mouse liver. Further, there can be carried out a methodfor producing a polypeptide possessing a ceramidase activityinexpensively and in a high purity by genetic engineering means.

In addition, the oligonucleotide probe or primer capable of specificallyhybridizing to the ceramidase gene of the present invention is useful insearching, detection, amplification and the like of the ceramidase geneof the present invention. The antibody or a fragment thereofspecifically binding to the polypeptide of the present invention isuseful in detection, identification, purification, and the like of aceramidase.

In addition, according to the method of controlling an amount of aceramide in a cell and/or in a tissue of the present invention, theamount of a ceramide in the cell and/or in the tissue can be controlledby introducing the ceramidase gene of the present invention or itsantisense nucleic acid into the cell and/or into the tissue. Therefore,such a method of control is useful in the treatment of a disease causedby an abnormal ceramide amount, for instance, but not being particularlylimited thereto, a treatment of a disease such as Farber's disease.

The present invention will be concretely explained by the examples,without by no means intending to limit the scope of the presentinvention to these examples.

EXAMPLE 1 Purification of Ceramidase

In 300 ml of a 0.25 M sucrose solution containing 1 mM EDTA(sucrose-EDTA solution) was homogenized 181 g of livers excised from 105Sea/ddY mice (produced by Seiwa Experimental Animals). The resultinghomogenate was centrifuged at 600×g for 10 minutes, and thereafter thesupernatant was collected. The resulting supernatant was furthercentrifuged at 2700×g for 30 minutes, and the precipitates werecollected.

The precipitated fraction was suspended in 480 ml of the sucrose-EDTAsolution to give a suspension. The resulting suspension was frozen at−80° C., and thereafter thawed under running water. Thisfreezing-thawing treatment was repeated twice. Thereafter, the treatedsuspension was centrifuged at 105000×g for 90 minutes, and supernatantand precipitates were each collected. The precipitates were subjected tothe treatments of freezing-thawing and centrifugation in the same manneras described above. The supernatant was collected, and combined with thepreviously obtained supernatant, to give 520 ml of a crude enzymeextract.

Two-hundred and sixty milliliters of the crude extract was applied onto100-ml DEAE-Sepharose FF (manufactured by Amersham Pharmacia) columnequilibrated with 20 mM phosphate buffer (pH 7.0) and then non-adsorbentsubstances were removed by wash. Thereafter, the elution was carried outwith the same buffer containing 1 M NaCl to collect 160 ml of an activefraction for ceramidase. The fraction was subsequently applied onto100-ml Phenyl-Sepharose FF (manufactured by Amersham Pharmacia) columnequilibrated with 20 mM Tris-hydrochloric acid buffer (pH 7.5)containing 1 M NaCl. Thereafter, the elution was carried out on aconcentration gradient of 2 to 0 M NaCl, and the elution was ethercarried out on a concentration gradient of 0 to 1% Polidocanol (tradename: Lubrol PX, manufactured by Nacalai Tesque Inc.). By thischromatography, 310 ml of an active fraction for ceramidase wascollected.

The resulting active fraction was applied onto 25-ml Chelating-SepharoseFF (manufactured by Amersham Pharmacia, Cu²⁺ bound type) columnequilibrated with 20 mM Tris-hydrochloric acid buffer (pH 7.5)containing 0.5 M NaCl and 0.1% Lubrol PX. The column was washed with thesame buffer and then with 20 mM Tris-hydrochloric acid buffer (pH 7.5)containing 0.1% Lubrol PX. Thereafter, the elution of the enzyme wascarried out with 20 mM Tris-hydrochloric acid buffer (pH 7.5) containing2 M NH₄Cl and 0.1% Lubrol PX. The eluted active fraction wasconcentrated by ultrafiltration, to give a concentrate. Next, the bufferin the concentrate was substituted with 20 mM Tris-hydrochloric acidbuffer (pH 7.5) containing 0.1% Lubrol PX, to give an enzyme solution.Thirty milliliters of the resulting enzyme solution was further appliedonto a porous HQ column (φ4.6×100 mm, manufactured by PerceptiveBiosystems) equilibrated with 20 mM Tris-hydrochloric acid buffer (pH7.5) containing 0.1% Lubrol PX. Subsequently, the elution was carriedout on a concentration gradient of 0 to 0.5 M NaCl, to give an activefraction. This active fraction was applied onto hydroxyapatite column(φ7.5×100 mm, manufactured by PENTAX equilibrated with 1 mM phosphatebuffer (pH 7.0) containing 0.2 M NaCl and 0.1% Lubrol PX. The ceramidasedid not adsorb to this column, and was collected on an effluent column.Thereafter, the fraction was subjected to gel filtration chromatographyby using Superose 200HR column (φ10×300 mm, manufactured by AmershamPharmacia) equilibrated with 20 mM Tris-hydrochloric acid buffer (pH7.5) containing 0.2 M NaCl and 0.3% Lubrol PX to give a purifiedceramidase. As a result of the above purification procedures, 58 mg of apurified ceramidase preparation was obtained.

Various characteristics of the resulting purified ceramidase preparationwere studied as described in the present specification. They were foundto be as follows:

action: hydrolyzing ceramide to generate a sphingoid and fatty acid;

substrate specificity: having a substrate specificity as shown in Table1 listed above;

optimum pH: as shown in FIG. 1, the optimum pH of this ceramidase beingfrom 7.0 to 8.0;

temperature stability: the reduced activity not being observed whentreated in 20 mM Tris-hydrochloric acid buffer (pH 7.5) containing 0.1%Polidocanol (trade name: Lubrol PX) at 37° C. for 24 hours, but theactivity being reduced to about 30% of that before the treatment whentreated for 60° C. for 1 hour; and

molecular weight: about 94 kDa on SDS-PAGE (under reducing conditions);and about 73 kDa on SDS-PAGE (under reducing conditions) in this enzymedigested by glycopeptidase F.

EXAMPLE 2 Partial Amino Acid Sequencing of Ceramidase

To 11 ml of a sample solution comprising 50 pmol ceramidase was added 20mM Tris-hydrochloric acid buffer (pH 7.5) containing 0.3% Lubrol PX. Theresulting sample solution was applied onto MonoQ PCl 6/5 column (100 μl,manufactured by Amersham Pharmacia). Subsequently, the ceramidasefraction adsorbed to the column was eluted with the same buffercontaining 0.4 M NaCl. By these procedures, the ceramidase-containingfraction was concentrated to a volume of 50 μl. The resultingconcentrate was subjected to SDS-polyacrylamide gel electrophoresis, andthe electrophoresed gel was stained with GelCode Blue Stain reagent(manufactured by Pierce). Next, the band corresponding to the ceramidasewas cut out. One-quarter of the cut-out gel fragment was subjected toextraction on 300 μl of 0.1 M Tris-hydrochloric acid buffer (pH 9.0)containing 0.1% SDS at 37° C. for 16 hours. Using the resulting extractas a sample, the N-terminal amino acid sequencing of the ceramidase wasperformed by using G1005A Protein Sequencing System (manufactured byHewlett-Packard) to determine an amino acid sequence N-term. SEQ ID NO:1 of Sequence Listing shows an amino acid sequence N-term.

In addition, the remaining three-quarter of the cut-out gel fragment waswashed with 1 ml of 0.5 M Tris-hydrochloric acid buffer (pH 9.2)/50%acetonitrile at 30° C. for 45 minutes. The gel was completely dried byusing nitrogen gas and centrifugal concentrator, and thereafter 10 μl of0.5 M Tris-hydrochloric acid buffer (pH 9.2) containing 0.5 μg ofProtease Lys-C (manufactured by Wako Pure Chemical Industries, Ltd.) wasadded thereto. Further, 0.1 M Tris-hydrochloric acid buffer (pH 9.2) wasadded until the gel was completely swollen, and the mixture was kept at37° C. for 16 hours to carry out the protease digestion of theceramidase. After the termination of the reaction, the procedures ofextracting with 150 μl of 0.1% trifluoroacetic acid/60% acetonitrile atroom temperature for 1 hour were repeated twice, and an extract wascollected. This extract was subjected to reversed phase chromatographyto purify the peptide fragment. The resulting peptide fragment wasanalyzed by Edman degradation method using the G1005A Protein SequencingSystem to determine partial amino acid sequences C-46 and C-53. SEQ IDNOs: 2 and 3 of Sequence Listing each shows an amino acid sequence forC-46 and C-53.

EXAMPLE 3 Amplification of DNA Fragment Comprising Ceramidase Gene byPCR Method

Sense mix primer 53-S1 and antisense mix primer 53-A3 were designed andsynthesized with a DNA synthesizer on the basis of the partial aminoacid sequence C53 of the ceramidase determined in Example 2. SEQ ID NOs:4 and 5 of Sequence Listing each show the nucleotide sequences of theprimers 53-S1 and 53-A3. PCR was carried out by using these primers. PCRwas carried out with mouse liver cDNA plasmid library (manufactured byTakara Shuzo Co., Ltd.) as a template. PCR was carried out by a reactionof 94° C., 9 minutes; thereafter 40 cycles of reaction, wherein onecycle comprises a process consisting of 94° C., 0.5 minutes—51° C., 0.5minutes—72° C., 1 minute; and further an incubation at 72° C. for 7minutes. By this PCR, a specific amplified DNA fragment of a size ofabout 70 bp was detected on agarose electrophoresis.

This amplified DNA was collected from the gel, and this DNA wasincorporated in pGEM-T easy vector (manufactured by Promega) toconstruct a recombinant plasmid. An insert DNA fragment of the plasmidwas subjected to a nucleotide sequencing. As a result, a partialnucleotide sequence P-1 of this fragment was determined. SEQ ID NO: 6 ofSequence Listing shows the nucleotide sequence of P-1. The sequence is asequence corresponding to the partial amino acid sequence C-53 of theceramidase determined in Example 2. It has been confirmed that a part ofthe desired ceramidase gene could be obtained.

Antisense primers MA1 and MA2 were designed and synthesized on the basisof the nucleotide sequence of P-1. SEQ ID NOs: 7 and 8 of SequenceListing each show the nucleotide sequences of the primers MA1 and MA2.In addition, sense primers T7in and T7out were designed and synthesizedon the basis of the nucleotide sequence of vector pAP3neo used in theconstruction of the mouse liver cDNA plasmid library. SEQ ID NOs: 9 and10 of Sequence Listing each show the nucleotide sequences of the primersT7in and T7out. Nested PCR was carried out by using these primers withthe mouse liver cDNA plasmid library as a template. A 1st PCR wascarried out by using the sense primer T7out and the antisense primer MA2by a reaction at 94° C., 9 minutes; thereafter 40 cycles of reaction,wherein one cycle comprises a process consisting of 94° C., 0.5 minutes—51° C., 0.5 minutes—72° C., 2 minutes; and further an incubation at 72°C. for 7 minutes. A 2nd PCR was carried out under the same conditions asthe 1st PCR except for using the sense primer T7in and the antisenseprimer MA1 with a reaction mixture of the 1st PCR as a template.Consequently, an amplified DNA fragment of a size of 335 bp wasobtained. This DNA fragment was used as a probe for colony hybridizationdescribed below.

EXAMPLE 4 Cloning of Ceramidase Gene

A transformant resulting from introducing the mouse liver cDNA plasmidlibrary was inoculated to a nylon filter (trade name Hybond-N⁺,manufactured by Amersham Pharmacia) on an LB agar medium platecontaining 100 μg/ml ampicillin, and about 30000 colonies were formedper one plate of 9.5×13.5 cm to prepare a master filter. A replica ofthis filter was prepared, and the resulting replica filter wasrespectively treated for 5 minutes on a filter paper immersed in a 10%SDS solution; 5 minutes on a filter paper immersed in a solutioncontaining 0.5 M NaOH and 1.5 M NaCl (denaturation); 5 minutes on afilter paper immersed in 0.5 M Tris-hydrochloric acid buffer (pH 7.5)containing 3 M NaCl (neutralization); and 5 minutes on a filter paperimmersed in 2×SSC solutions. Thereafter, the filter was rinsed with2×SSC solution. This filter was air-dried, and thereafter DNA wasimmobilized on a filter by ultraviolet ray irradiation to be used as afilter for colony hybridization.

As the probe for the hybridization, one prepared by ³²P-labeling 0.1 μgequivalent of the amplified DNA fragment obtained in Example 3 by usinga DNA labeling kit, Ready To Go (manufactured by Pharmacia) inaccordance with the protocol attached to the same kit. Theabove-mentioned filter was placed in a hybri-bag. The pre-hybridizationwas carried out at 60° C. for 1 hour in a hybridization solution(composition: 7% PEG6000, 10% SDS solution), and thereafter theabove-mentioned labeled probe was added to the mixture so as to have aconcentration of 0.006 pmol/ml, and the hybridization was carried outovernight at 60° C. Next the filter was washed three times each for 15minutes at 60° C. in the washing liquid (2×SSC, 0.1% SDS) previouslyheated to 60° C. After excess water was removed from the filter, andthereafter the filter was photosensitized on an imaging platemanufactured by Fuji Photo Film for 20 minutes. Thereafter, a signal wasdetected with BAS1000 imaging analyzer (manufactured by Fuji PhotoFilm). Subsequently, colonies were collected on a master filtercorresponding to a positive signal obtained by these procedures (firstscreening).

The collected colonies were suspended in an LB medium containing 100μg/ml ampicillin, and thereafter the suspension was spread over thenylon filter on an LB agar medium plate of 9.5×13.5 cm containing 100μg/ml ampicillin, and about 200 to about 1000 colonies were formed perone sheet to prepare a master filter. This filter was subjected toscreening of a positive clone in the same manner as the first screening,and a third screening was further carried out by the same procedures. Asa result of the third screening, the positive clone which was deduced tocontain the ceramidase gene could be isolated.

A plasmid was prepared from this positive clone, and this plasmid wasnamed plasmid pLCDase. The plasmid was digested with various kinds ofrestriction enzymes or a combination of plural restriction enzymes.Thereafter, each of the formed DNA fragments was subcloned, and itsnucleotide sequence was analyzed By the above procedures, an entirenucleotide sequence of the DNA fragment inserted in the plasmid pLCDasewas determined. The sequence is shown in SEQ ID NO: 11 of SequenceListing. In addition, the nucleotide sequence of the open reading frame(ORF) found in the sequence and the amino acid sequence of thepolypeptide encoded thereby are each shown in SEQ ID NOs: 12 and 13 ofSequence Listing. Further, the restriction enzyme map of theabove-mentioned DNA fragment and the position of the open reading framecontained in the DNA fragment are shown in FIG. 2.

As a result of analysis of the nucleotide sequence of theabove-mentioned ORF, there were confirmed that the nucleotide sequencecomprises a nucleotide sequence encoding a partial amino acid sequenceof the ceramidase elucidated by Example 2, and that this ORF encodesceramidase. In addition, when the amino acid sequence of the ceramidaseencoding the ORF was compared with the amino acid sequence of theceramidase shown in SEQ ID NO: 1 of Sequence Listing, it was shown thatthe ceramidase purified from the mouse liver lacked the peptide of theN-terminal portion of the polypeptides encoded by the above-mentionedORF. In other words, it was shown that the ceramidase was converted to amature enzyme by going through the processing in which its N-terminalportion was removed after the translation. SEQ ID NO: 14 of SequenceListing shows an amino acid sequence of the mature ceramidase, and SEQID NO: 15 of Sequence Listing shows a nucleotide sequence of the matureceramidase, respectively.

EXAMPLE 5 Expression of Ceramidase Gene

To CHO cells cultured in a 10% FCS-containing α-MEM medium in a dishhaving a diameter of 35 mm (3×10⁵ cells/dish) were added 1 μg of theplasmid pLCDase obtained in Example 4 and 5 μl of lipofectamine(manufactured by Life Technologies), thereby transducing a ceramidasegene into CHO cells. The cells were cultured at 37° C. for 24 hours, andthereafter suspended in 100 μl of 10 mM Tris-hydrochloric acid (pH 7.5)containing 0.1% Triton X-100, and the cells were disrupted. Thedetermination of the activity on the ceramidase of which substrate wasthe above-mentioned C12-NBD-ceramide was made for the resulting solutioncontaining disrupted cells. As a result, it was confirmed that theceramidase in the cells intensively expressed about 1000 times more thanthat of the control cells in which pLCDase was not transduced.

Further, the amount of ceramide in the cells was determined inaccordance with the method described in Analytical Biochemistry, 244,291-300 (1997). As a result it was confirmed that thosepLCDase-transduced cells had a significantly reduced amount of ceramide,as compared to the control cells into which pLCDase was not transduced.

EXAMPLE 6 Cloning of Ceramidase Gene from Mouse Brain

A nitrocellulose membrane (Schleicher & Schuell, PROTRAN BA85 0.45 mmbeing used with a diameter of 82 mm) was placed on an LB agar mediumplate containing 100 μg/ml ampicillin, and the mouse brain cDNA library(LIFE TECHNOLOGIES, SUPERSCRIPT Mouse Brain cDNA Library) was spread oneach of 10 plates, so as to be about 200000 colonies per one plate, andcultured at 37° C. for 10 hours. E. coli grown on the nitrocellulosemembrane was transferred to a nylon membrane [PALL Gelman Laboratory,biodyne A diameter 82 mm (1.2 mm)], and each nylon membrane was placedon an ampicillin plate, and thereafter cultured at 37° C. for 3 hours.The nitrocellulose membrane was stored at 4° C. as a master filter, andthe nylon membrane was placed on chloramphenicol plate, and cultured at37° C. for 16 hours. Colonies were transferred to a fresh nylon membranefrom the nylon membrane, and front and back sides of the membrane wereeach treated for 5 minutes with 1 ml of a denaturation solution (0.5 MNaOH/1.5 M NaCl) in a state where each pair of nylon membranes wasoverlaid. Similarly, the nylon membranes were treated with 1 ml of aneutralization solution [0.5 M Tris-HCl (pH 7.4)/1.5 M NaCl] for 5minutes. The nylon membrane was peeled of, and the treated membrane wasair-dried, and thereafter baked at 80° C. for 2 hours. Thereafter, thebaked membrane was shaken with 200 ml of a pre-rinsing liquid[5×SSC/0.5% SDS/1 ml EDTA (pH 8.0)], and disrupted E. coli residues werewiped off, and washed with 2×SCC. The hybridization was carried out with40 ml of a hybridization solution [0.5 M Church phosphate buffer/7%SDS/1 mM EDTA] at 65° C. for 2 hours, and thereafter the hybridizationwas carried out at 65° C. for 16 hours in 40 ml of the hybridizationsolution containing the denatured probe. As the probe, an EcoRI-EcoRIfragment of a size of 2.7 kbp of the plasmid a pAPLCD carrying mouseceramidase gene was used. After the termination of the hybridization,washing with 100 ml of a washing liquid (40 mM Church phosphatebuffer/1% SDS) at 65° C. for 15 minutes was carried out twice. Further,washing with 100 ml of a high stringent washing liquid (0.2×SSC/0.1%SDS) at 65° C. for 15 minutes was carried out. The membrane wasair-dried, and thereafter exposed on an IP-plate for 1 hour, andanalyzed with BAS 1500. The positive part of the nitrocellulose membranewas cut out in a diameter of about 6 mm, and suspended in 1 ml of the LBmedium. Thereafter, a 200 ml sample prepared by diluting the positivepart 4000-folds was spread over an ampicillin-containing LB plate, and a2nd screening was carried out.

Similarly, a 200 ml sample prepared by diluting the positive part10000-folds was spread over an ampicillin-containing LB plate, toprepare a library. A 3rd screening was carried out by using thislibrary. The isolated clone (pSBCD) was subcloned, and thereafter thenucleotide sequence was determined by a conventional method. Thissequence is shown in SEQ ID NO: 16.

In the above-mentioned sequence, an ORF having the identical sequence asthe ceramidase gene derived from mouse liver described in Example 4 wasfound. Incidentally, the sequences of 5′ non-translational region and 3′non-translational region were different from those derived from themouse liver. In addition, the isolated plasmid pSBCD was transduced intoCHO cells in the same manner as in Example 5. As a result, it was foundthat the ceramidase was expressed in the cells.

EXAMPLE 7 Genomic Cloning of Human Ceramidase Gene

Sense primer U1107 having the sequence of SEQ ID NO: 17 of SequenceListing and antisense primer L1311 having the sequence of SEQ ID NO: 18of Sequence Listing were synthesized on the basis of the sequence of theceramidase gene derived from mouse liver determined in Example 4. Thesequence of U1107 primer corresponds to a sequence consisting of basenos.: 1107-1130 of SEQ ID NO: 12 of Sequence Listing, and the sequenceof L1311 primer corresponds to a nucleotide sequence complementary to asequence consisting of base nos.: 1311-1334 of SEQ ID NO: 12 of SequenceListing.

Genomic DNA was purified from human hepatoma cell Huh7 by a conventionalmethod PCR was carried out by using the U1107 primer and the L1311primer with 625 ng of the resulting genomic DNA as a template. PCR wascarried out by a reaction at 94° C., 9 minutes; thereafter 40 cycles ofreaction, wherein one cycle comprises a process consisting of 94° C.,0.5 minutes—55° C., 0.5 minutes—72° C., 3 minutes; and further anincubation at 72° C. for 7 minutes. Thereafter, the resulting reactionproduct was subjected to agarose electrophoresis. As a result, it wasconfirmed that a DNA fragment of a size of about 2 kbp was amplified bythe PCR.

This DNA fragment was collected from the gel by using Sephaglas(manufactured by Pharmacia), and the resulting fragment was incorporatedin pGEM-T easy vector (manufactured by Promega) to construct arecombinant plasmid. Next, the nucleotide sequence of the DNA fragmentinsert of the resulting plasmid was determined. The above-mentionedsequence was a sequence corresponding to 96289-98478 of the AccessionNo. AC012131 Complement registered in the GenBank Data Base. Inaddition, the amino acid sequence encoded by the above-mentionedsequence was analyzed. As a result, a region encoding an amino acidsequence showing homology to a region of the amino acid nos.: 370-444 ofthe amino acid sequence of the ceramidase derived from mouse liver ofSEQ ID NO: 4 of Sequence Listing and a region encoding an amino acidsequence showing homology were found.

Incidentally, AC012131 also has homology with the ceramidase genederived from mouse liver determined in Example 4.

SEQUENCE LISTING FREE TEXT

In the amino acid sequence of SEQ ID NO: 1, each of Xaa in the aminoacid numbers: 7, 9 and 13 stands for an unknown amino acid.

SEQ ID NO: 4 is a sequence of synthetic oligonucleotide primers. In theabove-mentioned sequence, each of n in the base numbers: 6, 9 and 15stands for G, A, T or C.

SEQ ID NO: 5 is a sequence of synthetic oligonucleotide primers. In theabove-mentioned sequence, each of n in the base numbers: 3, 6 and 15stands for G, A, T or C.

SEQ ID NO: 7 is a sequence of synthetic oligonucleotide primers.

SEQ ID NO: 8 is a sequence of synthetic oligonucleotide primers.

SEQ ID NO: 9 is a sequence of synthetic oligonucleotide primers.

SEQ ID NO: 10 is a sequence of synthetic oligonucleotide primers.

SEQ ID NO: 11 is a sequence of synthetic oligonucleotide primers.

SEQ ID NO: 17 is a sequence of synthetic oligonucleotide primers.

SEQ ID NO: 18 is a sequence of synthetic oligonucleotide primers.

INDUSTRIAL APPLICABILITY

According to the present invention, a gene encoding a neutral/alkalineceramidase derived from a mammal is provided, and a genetic engineeringmethod for producing a ceramidase using the gene. Also, theoligonucleotide probe and primer of the pre sent invention are usefulfor detection of the above-mention and can be applied to studies on invivo ceramide metabolism. Further, according to the present invention,an antisense nucleic acid (DNA, RNA) of the gene of the presentinvention is provided. The gene and its antisense nucleic acid areuseful in the control of the ceramidase activity and in the regulationsof the in vivo ceramide metabolism system. Therefore, a method ofregulating an amount of ceramide which can be applied to treatments ofdiseases caused by the abnormal amount of ceramide is provided.

18 1 21 PRT Mus sp. MISC_FEATURE (1)..(21) any Xaa = any amino acid,unknown, or other 1 Phe Ser Gly Tyr Tyr Ile Xaa Val Xaa Arg Ala Asp XaaThr Gly Lys 1 5 10 15 Val Asn Asp Ile Asn 20 2 10 PRT Mus sp.MISC_FEATURE (1)..(10) any Xaa = any amino acid, unknown, or other 2 AlaIle Ala Thr Asp Thr Val Ala Xaa Met 1 5 10 3 35 PRT Mus sp. MISC_FEATURE(1)..(35) any Xaa = any amino acid, unknown, or other 3 Gly Tyr Leu ProGly Gln Gly Pro Phe Val Asn Gly Phe Ala Ser Ser 1 5 10 15 Asn Leu GlyAsp Val Ser Pro Asn Ile Leu Gly Pro Xaa Xaa Val Asn 20 25 30 Thr Gly Glu35 4 17 DNA Artificial Sequence Synthetic oligonucleotide primer 53-S1directed to gene derived from Mus sp. liver 4 carggnccnt tygtngc 17 5 17DNA Artificial Sequence Synthetic oligonucleotide primer 53-A3 directedto gene derived from Mus sp. liver 5 ggnccnagda trttngg 17 6 38 DNA Mussp. 6 gcaggctttg cttcatcaaa tctcggagac gtgtcacc 38 7 19 DNA ArtificialSequence Synthetic oligonucleotide primer MA1 directed to gene derivedfrom Mus sp. liver 7 ttgatgaagc aaagcctgc 19 8 19 DNA ArtificialSequence Synthetic oligonucleotide primer MA2 directed to gene derivedfrom Mus sp. liver 8 ggtgacacgt ctccgagat 19 9 20 DNA ArtificialSequence Synthetic oligonucleotide primer T7in directed to gene derivedfrom Mus sp. liver 9 taatacgact cactataggg 20 10 17 DNA ArtificialSequence Synthetic oligonucleotide primer T7out directed to gene derivedfrom Mus sp. liver 10 tctgctctaa aagctgc 17 11 3108 DNA Mus sp. 11cctgcgccac ttctctctcc cggctcaatc gcggagcctt ttctctcccc cgtctcgccg 60ctgccgccat ctccacccct gcctgcccca ggggtctgtg gacgcccggg cagagagcaa 120gcaccgagct gggcctgctg gagaccggag accagcggcc cgcccgcccg cccgctgcga 180gcctcctgag cagctccgga acagcttact ttctgtttcc atctctttcg gaccgggttg 240gcctctccaa aagccacttc tcctaactct tatcaaggtt caaaggctaa aggtctgtac 300acatgagtgc tggtgtgctt agaggcatcg ggtccctttc agctggagtt gcagtacttg 360tgagtgccat ggaatccaaa ttcggcaaga gatacaatct aaactctcaa ctactccaga 420ttcaaggttc acctcacttt ctggttacca aaggagcttt gcggggccgc tctgacatcc 480agtagatttg gaaacacatt gagaaatcag cctgagcaac ctgcaaggca caaggcacaa 540gattctgcat ggttatttgc tctcccagga ggtgaacact tgttttgatt cacagagtca 600gggttgagat gcccagttgt tcctcatctt ggctcagaag aagcacctag gaataaaagc 660tctaagctgg tattaagtag aatgggctta aagtccacta caggaaacaa cagctagtga 720cagaaatggc aaagcgaacc ttctccacct tggaggcatt cctcattttc cttctggtaa 780taatgacagt catcacagtg gcccttctca ccctcttgtt tgttaccagt gggaccattg 840aaaaccacaa agattcagga aatcactggt tttcaaccac tctgggctcc acgacaaccc 900agccccctcc aattacacag actccaaact tcccttcatt tcggaacttc agtggctact 960acattggcgt tgggagagcg gattgcacag gacaagtgtc agatatcaat ttgatgggct 1020atggcaaaaa tggccagaat gcacggggtc tcctcaccag gctgttcagc cgtgctttta 1080tcttggcgga tccagatggg tcaaatcgaa tggcatttgt gagcgtggaa ctatgtatga 1140tttcccaacg actgaggttg gaggtcctga agagactaga gagtaaatat ggctctctgt 1200atcgaagaga caatgttatc ctgagtgcca ttcacacaca ctctggccca gcagggtttt 1260tccaatatac actctatata ctcgccagcg agggattcag caaccggacc tttcagtaca 1320tagtctctgg gatcatgaag agcattgata tagctcacac aaatcttaaa ccaggcaaaa 1380tctttatcaa caaaggaaat gttgctaatg tgcagatcaa ccgaagcccc tcctcttacc 1440ttctgaatcc acagtcagag agagcaaggt attcttcaaa cacagacaag gaaatgctgg 1500tcttgaaact ggtggatttg aatggagaag acttgggtct tatcagctgg tttgccatcc 1560accccgtgag catgaacaat agcaaccact ttgtgaatag tgacaatatg ggctatgcgg 1620cttacctttt tgagcaagaa aagaacaaag gctatctgcc tggacaggga ccgtttgtag 1680caggctttgc ttcatcaaat ctcggagacg tgtcacccaa cattcttggc ccgcattgtg 1740tcaacacagg ggagtcttgt gacaacgaca agagcacctg tcccaacggt gggcctagca 1800tgtgcatggc cagcggacct ggacaagaca tgtttgagag cacacacatt ataggacgga 1860tcatctatca gaaggccaag gagctgtatg cctctgcctc ccaggaggtg accggcccag 1920tgcttgcagc tcaccagtgg gtgaacatga cagatgtgag cgtccagctc aatgccacac 1980acacagtgaa gacgtgtaaa cctgccctgg gctacagttt tgccgcaggc acaattgatg 2040gagtttcggg cctcaatatt acacagggaa ctacggaagg ggatccattc tgggacactc 2100ttcgggacca gctcttggga aaaccatctg aagagattgt agagtgtcag aaacccaaac 2160caatcctgct tcacagtgga gagctgacga taccacatcc ttggcaacca gatattgttg 2220atgttcagat tgttaccgtt gggtccttgg ccatagctgc tatccctggg gaattaacaa 2280ccatgtcggg acgaagattt cgtgaggcaa ttaaaaaaga atttgcactt tatgggatga 2340aggatatgac cgttgttatc gcaggtctaa gcaatgttta tacacattac attaccacat 2400atgaagaata ccaggctcag cggtacgagg cagcatctac aatctatgga ccacacaccc 2460tgtctgcata catccaactc ttcagagacc ttgctaaggc aattgctacg gacacagtag 2520ccaacatgag cagtggtccc gagcctccat tcttcaaaaa tctaatagct tcacttattc 2580ctaatattgc ggatagagca ccaattggca aacattttgg ggatgtcttg cagccagcaa 2640aacctgaata cagagtggga gaagtggttg aagttatatt tgtaggcgct aacccaaaga 2700attcagcaga gaaccagacc catcaaacct tcctcactgt ggagaaatac gaggactctg 2760tagctgactg gcagataatg tataacgatg cctcctggga gacgaggttt tattggcaca 2820aaggaatact gggtctgagc aatgcaacaa tatactggca tattccagat actgcctacc 2880ctggaatcta cagaataaga tattttggac acaatcggaa gcaggaactt ctgaaacccg 2940ctgtcatact agcatttgaa ggaatttctt ctccttttga agttgtcact acttagtgaa 3000aagttgacag atattgaaga aaagcttttc tctgtgcaca ttatagagtg aattcacaaa 3060aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 3108 12 2271 DNAMus sp. 12 atggcaaagc gaaccttctc caccttggag gcattcctca ttttccttctggtaataatg 60 acagtcatca cagtggccct tctcaccctc ttgtttgtta ccagtgggaccattgaaaac 120 cacaaagatt caggaaatca ctggttttca accactctgg gctccacgacaacccagccc 180 cctccaatta cacagactcc aaacttccct tcatttcgga acttcagtggctactacatt 240 ggcgttggga gagcggattg cacaggacaa gtgtcagata tcaatttgatgggctatggc 300 aaaaatggcc agaatgcacg gggtctcctc accaggctgt tcagccgtgcttttatcttg 360 gcggatccag atgggtcaaa tcgaatggca tttgtgagcg tggaactatgtatgatttcc 420 caacgactga ggttggaggt cctgaagaga ctagagagta aatatggctctctgtatcga 480 agagacaatg ttatcctgag tgccattcac acacactctg gcccagcagggtttttccaa 540 tatacactct atatactcgc cagcgaggga ttcagcaacc ggacctttcagtacatagtc 600 tctgggatca tgaagagcat tgatatagct cacacaaatc ttaaaccaggcaaaatcttt 660 atcaacaaag gaaatgttgc taatgtgcag atcaaccgaa gcccctcctcttaccttctg 720 aatccacagt cagagagagc aaggtattct tcaaacacag acaaggaaatgctggtcttg 780 aaactggtgg atttgaatgg agaagacttg ggtcttatca gctggtttgccatccacccc 840 gtgagcatga acaatagcaa ccactttgtg aatagtgaca atatgggctatgcggcttac 900 ctttttgagc aagaaaagaa caaaggctat ctgcctggac agggaccgtttgtagcaggc 960 tttgcttcat caaatctcgg agacgtgtca cccaacattc ttggcccgcattgtgtcaac 1020 acaggggagt cttgtgacaa cgacaagagc acctgtccca acggtgggcctagcatgtgc 1080 atggccagcg gacctggaca agacatgttt gagagcacac acattataggacggatcatc 1140 tatcagaagg ccaaggagct gtatgcctct gcctcccagg aggtgaccggcccagtgctt 1200 gcagctcacc agtgggtgaa catgacagat gtgagcgtcc agctcaatgccacacacaca 1260 gtgaagacgt gtaaacctgc cctgggctac agttttgccg caggcacaattgatggagtt 1320 tcgggcctca atattacaca gggaactacg gaaggggatc cattctgggacactcttcgg 1380 gaccagctct tgggaaaacc atctgaagag attgtagagt gtcagaaacccaaaccaatc 1440 ctgcttcaca gtggagagct gacgatacca catccttggc aaccagatattgttgatgtt 1500 cagattgtta ccgttgggtc cttggccata gctgctatcc ctggggaattaacaaccatg 1560 tcgggacgaa gatttcgtga ggcaattaaa aaagaatttg cactttatgggatgaaggat 1620 atgaccgttg ttatcgcagg tctaagcaat gtttatacac attacattaccacatatgaa 1680 gaataccagg ctcagcggta cgaggcagca tctacaatct atggaccacacaccctgtct 1740 gcatacatcc aactcttcag agaccttgct aaggcaattg ctacggacacagtagccaac 1800 atgagcagtg gtcccgagcc tccattcttc aaaaatctaa tagcttcacttattcctaat 1860 attgcggata gagcaccaat tggcaaacat tttggggatg tcttgcagccagcaaaacct 1920 gaatacagag tgggagaagt ggttgaagtt atatttgtag gcgctaacccaaagaattca 1980 gcagagaacc agacccatca aaccttcctc actgtggaga aatacgaggactctgtagct 2040 gactggcaga taatgtataa cgatgcctcc tgggagacga ggttttattggcacaaagga 2100 atactgggtc tgagcaatgc aacaatatac tggcatattc cagatactgcctaccctgga 2160 atctacagaa taagatattt tggacacaat cggaagcagg aacttctgaaacccgctgtc 2220 atactagcat ttgaaggaat ttcttctcct tttgaagttg tcactactta g2271 13 756 PRT Mus sp. 13 Met Ala Lys Arg Thr Phe Ser Thr Leu Glu AlaPhe Leu Ile Phe Leu 1 5 10 15 Leu Val Ile Met Thr Val Ile Thr Val AlaLeu Leu Thr Leu Leu Phe 20 25 30 Val Thr Ser Gly Thr Ile Glu Asn His LysAsp Ser Gly Asn His Trp 35 40 45 Phe Ser Thr Thr Leu Gly Ser Thr Thr ThrGln Pro Pro Pro Ile Thr 50 55 60 Gln Thr Pro Asn Phe Pro Ser Phe Arg AsnPhe Ser Gly Tyr Tyr Ile 65 70 75 80 Gly Val Gly Arg Ala Asp Cys Thr GlyGln Val Ser Asp Ile Asn Leu 85 90 95 Met Gly Tyr Gly Lys Asn Gly Gln AsnAla Arg Gly Leu Leu Thr Arg 100 105 110 Leu Phe Ser Arg Ala Phe Ile LeuAla Asp Pro Asp Gly Ser Asn Arg 115 120 125 Met Ala Phe Val Ser Val GluLeu Cys Met Ile Ser Gln Arg Leu Arg 130 135 140 Leu Glu Val Leu Lys ArgLeu Glu Ser Lys Tyr Gly Ser Leu Tyr Arg 145 150 155 160 Arg Asp Asn ValIle Leu Ser Ala Ile His Thr His Ser Gly Pro Ala 165 170 175 Gly Phe PheGln Tyr Thr Leu Tyr Ile Leu Ala Ser Glu Gly Phe Ser 180 185 190 Asn ArgThr Phe Gln Tyr Ile Val Ser Gly Ile Met Lys Ser Ile Asp 195 200 205 IleAla His Thr Asn Leu Lys Pro Gly Lys Ile Phe Ile Asn Lys Gly 210 215 220Asn Val Ala Asn Val Gln Ile Asn Arg Ser Pro Ser Ser Tyr Leu Leu 225 230235 240 Asn Pro Gln Ser Glu Arg Ala Arg Tyr Ser Ser Asn Thr Asp Lys Glu245 250 255 Met Leu Val Leu Lys Leu Val Asp Leu Asn Gly Glu Asp Leu GlyLeu 260 265 270 Ile Ser Trp Phe Ala Ile His Pro Val Ser Met Asn Asn SerAsn His 275 280 285 Phe Val Asn Ser Asp Asn Met Gly Tyr Ala Ala Tyr LeuPhe Glu Gln 290 295 300 Glu Lys Asn Lys Gly Tyr Leu Pro Gly Gln Gly ProPhe Val Ala Gly 305 310 315 320 Phe Ala Ser Ser Asn Leu Gly Asp Val SerPro Asn Ile Leu Gly Pro 325 330 335 His Cys Val Asn Thr Gly Glu Ser CysAsp Asn Asp Lys Ser Thr Cys 340 345 350 Pro Asn Gly Gly Pro Ser Met CysMet Ala Ser Gly Pro Gly Gln Asp 355 360 365 Met Phe Glu Ser Thr His IleIle Gly Arg Ile Ile Tyr Gln Lys Ala 370 375 380 Lys Glu Leu Tyr Ala SerAla Ser Gln Glu Val Thr Gly Pro Val Leu 385 390 395 400 Ala Ala His GlnTrp Val Asn Met Thr Asp Val Ser Val Gln Leu Asn 405 410 415 Ala Thr HisThr Val Lys Thr Cys Lys Pro Ala Leu Gly Tyr Ser Phe 420 425 430 Ala AlaGly Thr Ile Asp Gly Val Ser Gly Leu Asn Ile Thr Gln Gly 435 440 445 ThrThr Glu Gly Asp Pro Phe Trp Asp Thr Leu Arg Asp Gln Leu Leu 450 455 460Gly Lys Pro Ser Glu Glu Ile Val Glu Cys Gln Lys Pro Lys Pro Ile 465 470475 480 Leu Leu His Ser Gly Glu Leu Thr Ile Pro His Pro Trp Gln Pro Asp485 490 495 Ile Val Asp Val Gln Ile Val Thr Val Gly Ser Leu Ala Ile AlaAla 500 505 510 Ile Pro Gly Glu Leu Thr Thr Met Ser Gly Arg Arg Phe ArgGlu Ala 515 520 525 Ile Lys Lys Glu Phe Ala Leu Tyr Gly Met Lys Asp MetThr Val Val 530 535 540 Ile Ala Gly Leu Ser Asn Val Tyr Thr His Tyr IleThr Thr Tyr Glu 545 550 555 560 Glu Tyr Gln Ala Gln Arg Tyr Glu Ala AlaSer Thr Ile Tyr Gly Pro 565 570 575 His Thr Leu Ser Ala Tyr Ile Gln LeuPhe Arg Asp Leu Ala Lys Ala 580 585 590 Ile Ala Thr Asp Thr Val Ala AsnMet Ser Ser Gly Pro Glu Pro Pro 595 600 605 Phe Phe Lys Asn Leu Ile AlaSer Leu Ile Pro Asn Ile Ala Asp Arg 610 615 620 Ala Pro Ile Gly Lys HisPhe Gly Asp Val Leu Gln Pro Ala Lys Pro 625 630 635 640 Glu Tyr Arg ValGly Glu Val Val Glu Val Ile Phe Val Gly Ala Asn 645 650 655 Pro Lys AsnSer Ala Glu Asn Gln Thr His Gln Thr Phe Leu Thr Val 660 665 670 Glu LysTyr Glu Asp Ser Val Ala Asp Trp Gln Ile Met Tyr Asn Asp 675 680 685 AlaSer Trp Glu Thr Arg Phe Tyr Trp His Lys Gly Ile Leu Gly Leu 690 695 700Ser Asn Ala Thr Ile Tyr Trp His Ile Pro Asp Thr Ala Tyr Pro Gly 705 710715 720 Ile Tyr Arg Ile Arg Tyr Phe Gly His Asn Arg Lys Gln Glu Leu Leu725 730 735 Lys Pro Ala Val Ile Leu Ala Phe Glu Gly Ile Ser Ser Pro PheGlu 740 745 750 Val Val Thr Thr 755 14 682 PRT Mus sp. 14 Phe Ser GlyTyr Tyr Ile Gly Val Gly Arg Ala Asp Cys Thr Gly Gln 1 5 10 15 Val SerAsp Ile Asn Leu Met Gly Tyr Gly Lys Asn Gly Gln Asn Ala 20 25 30 Arg GlyLeu Leu Thr Arg Leu Phe Ser Arg Ala Phe Ile Leu Ala Asp 35 40 45 Pro AspGly Ser Asn Arg Met Ala Phe Val Ser Val Glu Leu Cys Met 50 55 60 Ile SerGln Arg Leu Arg Leu Glu Val Leu Lys Arg Leu Glu Ser Lys 65 70 75 80 TyrGly Ser Leu Tyr Arg Arg Asp Asn Val Ile Leu Ser Ala Ile His 85 90 95 ThrHis Ser Gly Pro Ala Gly Phe Phe Gln Tyr Thr Leu Tyr Ile Leu 100 105 110Ala Ser Glu Gly Phe Ser Asn Arg Thr Phe Gln Tyr Ile Val Ser Gly 115 120125 Ile Met Lys Ser Ile Asp Ile Ala His Thr Asn Leu Lys Pro Gly Lys 130135 140 Ile Phe Ile Asn Lys Gly Asn Val Ala Asn Val Gln Ile Asn Arg Ser145 150 155 160 Pro Ser Ser Tyr Leu Leu Asn Pro Gln Ser Glu Arg Ala ArgTyr Ser 165 170 175 Ser Asn Thr Asp Lys Glu Met Leu Val Leu Lys Leu ValAsp Leu Asn 180 185 190 Gly Glu Asp Leu Gly Leu Ile Ser Trp Phe Ala IleHis Pro Val Ser 195 200 205 Met Asn Asn Ser Asn His Phe Val Asn Ser AspAsn Met Gly Tyr Ala 210 215 220 Ala Tyr Leu Phe Glu Gln Glu Lys Asn LysGly Tyr Leu Pro Gly Gln 225 230 235 240 Gly Pro Phe Val Ala Gly Phe AlaSer Ser Asn Leu Gly Asp Val Ser 245 250 255 Pro Asn Ile Leu Gly Pro HisCys Val Asn Thr Gly Glu Ser Cys Asp 260 265 270 Asn Asp Lys Ser Thr CysPro Asn Gly Gly Pro Ser Met Cys Met Ala 275 280 285 Ser Gly Pro Gly GlnAsp Met Phe Glu Ser Thr His Ile Ile Gly Arg 290 295 300 Ile Ile Tyr GlnLys Ala Lys Glu Leu Tyr Ala Ser Ala Ser Gln Glu 305 310 315 320 Val ThrGly Pro Val Leu Ala Ala His Gln Trp Val Asn Met Thr Asp 325 330 335 ValSer Val Gln Leu Asn Ala Thr His Thr Val Lys Thr Cys Lys Pro 340 345 350Ala Leu Gly Tyr Ser Phe Ala Ala Gly Thr Ile Asp Gly Val Ser Gly 355 360365 Leu Asn Ile Thr Gln Gly Thr Thr Glu Gly Asp Pro Phe Trp Asp Thr 370375 380 Leu Arg Asp Gln Leu Leu Gly Lys Pro Ser Glu Glu Ile Val Glu Cys385 390 395 400 Gln Lys Pro Lys Pro Ile Leu Leu His Ser Gly Glu Leu ThrIle Pro 405 410 415 His Pro Trp Gln Pro Asp Ile Val Asp Val Gln Ile ValThr Val Gly 420 425 430 Ser Leu Ala Ile Ala Ala Ile Pro Gly Glu Leu ThrThr Met Ser Gly 435 440 445 Arg Arg Phe Arg Glu Ala Ile Lys Lys Glu PheAla Leu Tyr Gly Met 450 455 460 Lys Asp Met Thr Val Val Ile Ala Gly LeuSer Asn Val Tyr Thr His 465 470 475 480 Tyr Ile Thr Thr Tyr Glu Glu TyrGln Ala Gln Arg Tyr Glu Ala Ala 485 490 495 Ser Thr Ile Tyr Gly Pro HisThr Leu Ser Ala Tyr Ile Gln Leu Phe 500 505 510 Arg Asp Leu Ala Lys AlaIle Ala Thr Asp Thr Val Ala Asn Met Ser 515 520 525 Ser Gly Pro Glu ProPro Phe Phe Lys Asn Leu Ile Ala Ser Leu Ile 530 535 540 Pro Asn Ile AlaAsp Arg Ala Pro Ile Gly Lys His Phe Gly Asp Val 545 550 555 560 Leu GlnPro Ala Lys Pro Glu Tyr Arg Val Gly Glu Val Val Glu Val 565 570 575 IlePhe Val Gly Ala Asn Pro Lys Asn Ser Ala Glu Asn Gln Thr His 580 585 590Gln Thr Phe Leu Thr Val Glu Lys Tyr Glu Asp Ser Val Ala Asp Trp 595 600605 Gln Ile Met Tyr Asn Asp Ala Ser Trp Glu Thr Arg Phe Tyr Trp His 610615 620 Lys Gly Ile Leu Gly Leu Ser Asn Ala Thr Ile Tyr Trp His Ile Pro625 630 635 640 Asp Thr Ala Tyr Pro Gly Ile Tyr Arg Ile Arg Tyr Phe GlyHis Asn 645 650 655 Arg Lys Gln Glu Leu Leu Lys Pro Ala Val Ile Leu AlaPhe Glu Gly 660 665 670 Ile Ser Ser Pro Phe Glu Val Val Thr Thr 675 68015 2049 DNA Mus sp. 15 ttcagtggct actacattgg cgttgggaga gcggattgcacaggacaagt gtcagatatc 60 aatttgatgg gctatggcaa aaatggccag aatgcacggggtctcctcac caggctgttc 120 agccgtgctt ttatcttggc ggatccagat gggtcaaatcgaatggcatt tgtgagcgtg 180 gaactatgta tgatttccca acgactgagg ttggaggtcctgaagagact agagagtaaa 240 tatggctctc tgtatcgaag agacaatgtt atcctgagtgccattcacac acactctggc 300 ccagcagggt ttttccaata tacactctat atactcgccagcgagggatt cagcaaccgg 360 acctttcagt acatagtctc tgggatcatg aagagcattgatatagctca cacaaatctt 420 aaaccaggca aaatctttat caacaaagga aatgttgctaatgtgcagat caaccgaagc 480 ccctcctctt accttctgaa tccacagtca gagagagcaaggtattcttc aaacacagac 540 aaggaaatgc tggtcttgaa actggtggat ttgaatggagaagacttggg tcttatcagc 600 tggtttgcca tccaccccgt gagcatgaac aatagcaaccactttgtgaa tagtgacaat 660 atgggctatg cggcttacct ttttgagcaa gaaaagaacaaaggctatct gcctggacag 720 ggaccgtttg tagcaggctt tgcttcatca aatctcggagacgtgtcacc caacattctt 780 ggcccgcatt gtgtcaacac aggggagtct tgtgacaacgacaagagcac ctgtcccaac 840 ggtgggccta gcatgtgcat ggccagcgga cctggacaagacatgtttga gagcacacac 900 attataggac ggatcatcta tcagaaggcc aaggagctgtatgcctctgc ctcccaggag 960 gtgaccggcc cagtgcttgc agctcaccag tgggtgaacatgacagatgt gagcgtccag 1020 ctcaatgcca cacacacagt gaagacgtgt aaacctgccctgggctacag ttttgccgca 1080 ggcacaattg atggagtttc gggcctcaat attacacagggaactacgga aggggatcca 1140 ttctgggaca ctcttcggga ccagctcttg ggaaaaccatctgaagagat tgtagagtgt 1200 cagaaaccca aaccaatcct gcttcacagt ggagagctgacgataccaca tccttggcaa 1260 ccagatattg ttgatgttca gattgttacc gttgggtccttggccatagc tgctatccct 1320 ggggaattaa caaccatgtc gggacgaaga tttcgtgaggcaattaaaaa agaatttgca 1380 ctttatggga tgaaggatat gaccgttgtt atcgcaggtctaagcaatgt ttatacacat 1440 tacattacca catatgaaga ataccaggct cagcggtacgaggcagcatc tacaatctat 1500 ggaccacaca ccctgtctgc atacatccaa ctcttcagagaccttgctaa ggcaattgct 1560 acggacacag tagccaacat gagcagtggt cccgagcctccattcttcaa aaatctaata 1620 gcttcactta ttcctaatat tgcggataga gcaccaattggcaaacattt tggggatgtc 1680 ttgcagccag caaaacctga atacagagtg ggagaagtggttgaagttat atttgtaggc 1740 gctaacccaa agaattcagc agagaaccag acccatcaaaccttcctcac tgtggagaaa 1800 tacgaggact ctgtagctga ctggcagata atgtataacgatgcctcctg ggagacgagg 1860 ttttattggc acaaaggaat actgggtctg agcaatgcaacaatatactg gcatattcca 1920 gatactgcct accctggaat ctacagaata agatattttggacacaatcg gaagcaggaa 1980 cttctgaaac ccgctgtcat actagcattt gaaggaatttcttctccttt tgaagttgtc 2040 actacttag 2049 16 4835 DNA Mus sp. 16cctgcagcgg tgttctgaag agccgggcag aggatacaca agcatcccag caggcactct 60ggtttgcccg tgaacgatag atatgcgggg gtttgaatga gcagctgcag cagcgggttt 120gggtctgtac acatgagtgc tggtgtgctt agaggcatcg ggtccctttc agctggagtt 180gcagtacttg tgagtgccat atttggaaac acattgagaa atcagcctga gcaacctgca 240aggcacaagg cacaagattc tgcatggtta tttgctctcc caggaggtga acacttgttt 300tgattaacag agtcagggtt gagatgccca gttgttcctc atcttggctc agaagaagca 360cctaggaata aaagctctaa gctggtatta agtagaatgg gcttaaagtc cactacagga 420aacaacagct agtgacagaa atggcaaagc gaaccttctc caccttggag gcattcctca 480ttttccttct ggtaataatg acagtcatca cagtggccct tctcaccctc ttgtttgtta 540ccagtgggac cattgaaaac cacaaagatt caggaaatca ctggttttca accactctgg 600gctccacgac aacccagccc cctccaatta cacagactcc aaacttccct tcatttcgga 660acttcagtgg ctactacatt ggcgttggga gagcagattg cacaggacaa gtgtcagata 720tcaatttgat gggctatggc aaaaatggcc agaatgcacg gggtctcctc accaggctgt 780tcagccgtgc ttttatcttg gcggatccag atgggtcaaa tcgaatggca tttgtgagcg 840tggaactatg tatgatttcc caacgactga ggttggaggt cctgaagaga ctagagagta 900aatatggctc tctgtatcga agagacaatg ttatcctgag tgccattcac acacactctg 960gcccagcagg gtttttccaa tatacactct atatactcgc cagcgaggga ttcagcaacc 1020ggacctttca gtacatagtc tctgggatca tgaagagcat tgatatagct cacacaaatc 1080ttaaaccagg caaaatcttt atcaacaaag gaaatgttgc taatgtgcag atcaaccgaa 1140gcccctcctc ttaccttctg aatccacagt cagagagagc aaggtattct tcaaacacag 1200acaaggaaat gctggtcttg aaactggtgg atttgaatgg agaagacttg ggtcttatca 1260gctggtttgc catccacccc gtgagcatga acaatagcaa ccactttgtg aatagtgaca 1320atatgggcta tgcggcttac ctttttgagc aagaaaagaa caaaggctat ctgcctggac 1380agggaccgtt tgtagcaggc tttgcttcat caaatctcgg agacgtgtca cccaacattc 1440ttggcccgca ttgtgtcaac acaggggagt cttgtgacaa cgacaagagc acctgtccca 1500acggtgggcc tagcatgtgc atggccagcg gacctggaca agacatgttt gagagcacac 1560acattatagg acggatcatc tatcagaagg ccaaggagct gtatgcctct gcctcccagg 1620aggtgaccgg cccagtgctt gcagctcacc agtgggtgaa catgacagat gtgagcgtcc 1680agctcaatgc cacacacaca gtgaagacgt gtaaacctgc cctgggctac agttttgccg 1740caggcacaat tgatggagtt tcgggcctca atattacaca gggaactacg gaaggggatc 1800cattctggga cactcttcgg gaccagctct tgggaaaacc atctgaagag attgtagagt 1860gtcagaaacc caaaccaatc ctgcttcaca gtggagagct gacgatacca catccttggc 1920aaccagatat tgttgatgtt cagattgtta ccgttgggtc cttggccata gctgctatcc 1980ctggggaatt aacaaccatg tcgggacgaa gatttcgtga ggcaattaaa aaagaatttg 2040cactttatgg gatgaaggat atgaccgttg ttatcgcagg tctaagcaat gtttatacac 2100attacattac cacatatgaa gaataccagg ctcagcggta cgaggcagca tctacaatct 2160atggaccaca caccctgtct gcatacatcc aactcttcag agaccttgct aaggcaattg 2220ctacggacac agtagccaac atgagcagtg gtcccgagcc tccattcttc aaaaatctaa 2280tagcttcact tattcctaat attgcggata gagcaccaat tggcaaacat tttggggatg 2340tcttgcagcc agcaaaacct gaatacagag tgggagaagt ggttgaagtt atatttgtag 2400gcgctaaccc aaagaattca gcagagaacc agacccatca aaccttcctc actgtggaga 2460aatacgagga ctctgtagct gactggcaga taatgtataa cgatgcctcc tgggagacga 2520ggttttattg gcacaaagga atactgggtc tgagcaatgc aacaatatac tggcatattc 2580cagatactgc ctaccctgga atctacagaa taagatattt tggacacaat cggaagcagg 2640aacttctgaa acccgctgtc atactagcat ttgaaggaat ttcttctcct tttgaagttg 2700tcactactta gtgaaaagtt gacagatatt gaagaaaagc ttttctctgt gcacattata 2760gagtgaattc acacaaatgt gaactgccag tttaatttct gtaattgtct ctgtttgggg 2820gacaggtcat ttattgctaa tgggacagag gtatgtgttt gtgttgttgt atgattatga 2880gtatgcatgc taacaggaag agagagggag gagggaggga ggagggggag ggagggaaga 2940aaggagggag agagagagtg agagaatgag agagagagtg agagagaaag agttattagt 3000gagcaagaga atatgagaga agggccactg acaaccaaat accttgtgat ctttatccta 3060aagcatgatt ttccttgaag ctctgtggtt gtttaagaga taattccctc taatatgaaa 3120tccctgaaat ataatgacag tatttgaaga tatgtgaata atgtttatcc tatttattta 3180tagacttact aaatgagaac actagagaac tttctagaag tcctctagaa tgatacttga 3240ttttacagag aggaaaagga gctttgattc tctttaggtt agaataaggt tagtatattt 3300ttccctagtc atatttacaa aataccatgt aactttacta caaatatttg agcccagcta 3360aaatataccc agaaaattag cataccagtt ttgttttgtt ttattttgtt tttgcatcca 3420aacaagcata gtccttctga taagtcactt tagaatggat ctgcctggct cagggttatt 3480gttcatgctc agatcatttc cgcaattacc tccagagtcc aactatgcga atgtcacttg 3540cagtgctttg atttatgcct tgtattcctc aaagtgtcct tatcctgcta agtcacacct 3600cttcctccca gcatttactc taaatgattt ttaatgtttt cgccaatcaa atgtacctca 3660cattacaaag ctttgccttg aatgtagatt tttaaaacaa aagtgttaag gctggaaatg 3720tagttatcaa agaggaagtt ttaaatgtat ctgttctttt atcagctact ccctccctca 3780tggctccctt gaatcactga atagttattt aaacccacat atccaatatg gtactcattc 3840ctgggtcttc acaattacag acatcatatc gaaatgattg ggctgacaat tcctttgaag 3900gacaaagtaa atatttaatg agaaatatag attctggaga ggcatttgaa aatcacaaat 3960gttacgcctc catttcctgt tttccaggct gggtgttctg atttgggagg aaagcagccc 4020caaataattt ttaaatatga atctgaaaat aatgttttag aaattatgat ctcgacagtc 4080taattaatga gaattgtctg aaagtcctag ctgcatttaa aattatgtaa gttaactaaa 4140gccaattttt gaaccccagt cataattgtg taggtaggta aaaagagcat tttaggagga 4200aaccgaactt catttcaaga ctgaatctgt tttaaaagaa caatagtggt aaggtaaatc 4260ttcatttatt tccctatggt ttacctattt aaacatcgaa gattgaatca aaaggcacct 4320ggagcatatt ttggtaactc catttcccac ttggtagttc tatggatgct aactgctgaa 4380gaataaactg atcgggattt tcaagggttg tgaacatgtc tcctgatggg aataccgtat 4440taagtataaa ggttcaaaat agttgatctc aaaactatac acacacacac aatatatata 4500tatatacaca cacacacatg tacacacaca cacacatgca catacacatg gtattgttta 4560aaatttattt ctcatgactt agaacaatat aaggattata caaggattca tttcccacca 4620tcattcctcc cagtgaagct tttctcaaag tctgagtagg agtttctcct ttctcactgg 4680taactatccc acagtggcca ttacatcact agtaatcggt gtgcccagcc ctgcatggaa 4740ataaatcaca gaaacataat ttcccagtag acttagtctc ttcaagcctg tgtgcttcta 4800gtgtataaaa tctgtaaaaa aaaaaaaaaa aaaaa 4835 17 24 DNA ArtificialSequence Synthetic oligonucleotide sense primer U1107 directed to genederived from Mus sp. liver 17 gtttgagagc acacacatta tagg 24 18 24 DNAArtificial Sequence Synthetic oligonucleotide antisense primer L1311directed to gene derived from Mus sp. liver 18 atattgaggc ccgaaactccatca 24

What is claimed is:
 1. An isolated nucleic acid comprising a nucleotidesequence selected from the group consisting of: (A) a nucleotidesequence encoding a polypeptide having the amino acid sequence of SEQ IDNO: 14, wherein the polypeptide possesses a ceramidase activity; (B) anucleotide sequence comprising SEQ ID NO: 15, wherein said nucleotidesequence encodes a polypeptide possessing a ceramidase activity; and (C)a nucleotide sequence which hybridizes to the complement of thenucleotide sequence of SEQ ID NO: 15 under stringent conditions, whereinsaid stringent conditions comprise 7% PEG 6000 containing 10% SDSsolution at 60° C., and washing three times with 2×SSC containing 0.1%SDS for 15 minutes at 60° C., wherein said nucleotide sequence encodes apolypeptide having activity of hydrolyzing any one of substancesselected from the group consisting of (i) N-Lauroylsphinqosine, (ii)N-Palmitoylsphingosine, (iii) N-Stearoylsphingosine, (iv)N-Palmitoylsphinganine, (v) N-Stearoylsphinganine, and (vi)12-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl)sphingosine.2. The nucleic acid according to claim 1, wherein the ceramidaseactivity of the polypeptide is detected by the following steps: (a)incubating an expression product in a reaction mixture comprising 550pmol of12-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl)sphingosineand 1.0% (W/V) sodium cholate in 20 μl of 25 mM Tris-hydrochloric acidbuffer, pH 7.5, at 37° C. for 30 minutes; and (b) detecting theformation of a 12-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoicacid in the reaction.
 3. The nucleic acid according to claim 1 or 2,wherein the polypeptide exhibits at least the following characteristics:(i) action of hydrolyzing ceramide to generate sphingoid and a fattyacid; (ii) substrate specificity of hydrolyzing N-acylsphingosine, butnot acting on galactosylceramide, sulfatide,Galb1-3GalNAcb1-4(NeuAca2-3)Galb1-4Glcb1-1′Cer (GM1a), andsphingomyelin; (iii) optimum pH from 7.0 to 8.0; and (iv) incubation in20 mM Tris-hydrochloric acid buffer, pH 7.5, containing 0.1% polidocanolat 37° C. for 24 hours does not decrease activity of said polypeptide,whereas incubation in 20 mM Tris-hydrochloric acid buffer, pH 7.5,containing 0.1% polidocanol at 60° C. for 1 hour decreases activity ofsaid polypeptide to about 30%.
 4. A recombinant DNA comprising thenucleic acid of claim
 1. 5. An expression vector comprising the nucleicacid of claim 1 or the recombinant DNA of claim
 4. 6. A transformed cellcomprising the expression vector of claim
 5. 7. A method for producing apolypeptide possessing a ceramidase activity, comprising the steps ofculturing the transformed cell of claim 6 under conditions appropriatefor expression of the polypeptide, and collecting a polypeptidepossessing a ceramidase activity from the resulting culture.
 8. Anisolated DNA which is complementary to the nucleic acid of claim
 1. 9.An isolated RNA which is complementary to the nucleic acid of claim 1.10. An expression vector comprising the DNA of claim
 8. 11. An isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO: 14, whereinsaid polypeptide possesses a ceramidase activity.
 12. An isolatedpolypeptide possessing a ceramidase activity, wherein said polypeptideis encoded by the nucleic acid of claim
 1. 13. The polypeptide accordingto claim 11 or 12, wherein the ceramidase activity is detected by thefollowing steps: (a) incubating an expression product in a reactionmixture comprising 550 pmol of12-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl)sphingosineand 1.0% (W/V) sodium cholate in 20 μl of 25 mM Tris-hydrochloric acidbuffer, pH 7.5, at 37° C. for 30 minutes; and (b) detecting theformation of a 12-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoicacid in the reaction.
 14. A method of controlling an amount of ceramidein a cell, comprising the step of introducing the nucleic acid of claim1 or a complementary nucleic acid thereof into the cell, therebycontrolling the amount of ceramide in the cell.